Comp. Biochem, Physiol.. 1976. Vol, 54B. pp. 339 to 342. Perflamon Press. Printed in Great Britain
DIFFERENCES IN MITOCHONDRIAL F U N C T I O N S FROM RIGHT A N D LEFT VENTRICULAR M Y O C A R D I U M OF FOUR MAMMALIAN SPECIES* LOUIS A. SORDAHL Division of Biochemistry, University of Texas Medical Branch, Galveston, TX 77550, U.S.A. (Received 22 July 1975)
Abstract--1. Mitochondria were isolated from the right and left ventricles of Canis familiaris, Felis catus, Oryctolagus cuniculus and Sus scrofa hearts. 2. A consistent 20-25~o lower rate of phosphorylating respiration (State 3) was found in right ventricular mitochondria compared to left in all four species. 3. Measurement of initial velocities of calcium uptake using dual-beam spectrophotometry revealed marked differences in the apparent KM and Vmaxvalues between the mitochondrial preparations from the four species. 4. The results indicate significant differences in mitochondrial respiratory activity between right and left myocardium and further suggest possible differences in the role of mitochondria in maintaining cellular calcium homeostasis in the various species' hearts.
INTRODUCTION
independently determined by Page & McCallister (1973) for normal rat left ventricle. Carafoli & Lehninger (1971) have surveyed mitochondrial calcium transport in a number of vertebrate and invertebrate tissues, including heart. These investigators (Carafoli & Lehninger, 1971) found no significant difference in mitochondrial calcium transport functions in all mammalian tissues studied. Recently, Braimbridge et al. (1973) demonstrated differences in enzyme levels of left ventricular free wall and papillary muscles in patients undergoing open-heart surgery. Several years ago this investigator observed a consistent difference in mitochondrial functions in right and left ventricular tissue obtained from cardiac transplant recipients (Lindenmayer et al., 1971). Although these hearts were in an abnormal state and a good deal of drug intervention preceded obtaining the tissues for subsequent biochemical analyses, this observation pointed out the possible differences in the functionality of mitochondria from various parts of the ventricular myocardium. Mitochondria not only constitute a significant portion of the total cytoplasmic volume of the myocardial cell, but under normal circumstances are the sole source of ATP for cellular processes. Energy production in the heart cell is directly related to the oxidative processes of mitochondria. Further, this organelle system of the heart may very well be involved in the movements of calcium related to excitation-contraction coupling in the myocardial cell. Evidence to date does not establish a direct role for mitochondria in the calcium fluxes related to myocardial contractility, but the possibility that mitochondria may play a role in maintaining the ionic homeostasis of the heart cell has been suggested by several investigators (Bygrave, 1967; Kiibler & Shinebourne, 1971 ; Borle, 1973). This report describes distinct and consistent differences in *Supported by grants from USPHS, 5 respiratory activity of mitochondria isolated from the SO1-RR-05427-12, HL 14828, USAF, AFOSR 74-2622 and right and left ventricular myocardium of four mammalian species. Determination of initial velocities of Texas Heart Association. 339
It is well known that the mammalian heart, at birth, is relatively equal in size with respect to the number of fibers in the right and left myocardium (Hort, 1971). During early post-natal development the left heart (ventricle) undergoes considerable enlargement relative to the right, yet the number of fibers on both sides remains constant (Hort, 1971). A number of studies have been reported (Alpert, 1971; Bajusz & Rona, 1972) on functional and structural changes of mammalian heart in experimentally induced cardiac pathologies. Specific changes in mitochondrial numbers, mass, volume and distribution have been shown in both right and left ventricular hypertrophy (Wollenberger & Schulze, 1961 ; Meerson, et al., 1964; Bishop & Cole, 1969). Using quantitative biometric techniques Anversa et aL (1971), Page & McCallister (1973); Goldstein et al. (1974) have demonstrated significant decreases in the mitochondria-myofibril ratio during left ventricular hypertrophy induced by aortic constriction. The above studies indicate marked ultrastructural changes can occur in ventricular myocardium under pathologic circumstances. Alterations in mitochondrial functions during cardiac hypertrophy and failure have also been observed (Sordahl et aL, 1971). However, very little has been reported on comparative ultrastructur¢ and function of mitochondria in normal myocardium. Laguens (1971) has reported 20~o fewer mitochondria in the right ventricle of normal rat myocardium compared to left. Employing quantitative hiometric techniques, Laguens (1971) found 36.6~o of the cytoplasmic volume occupied by mitochondria in the left ventricle and 29.3?/0 in the right. This value for left ventricular mitochondrial volumetric density is in close agreement to that of 35.8~o
340 calcium marked calcium various
Louis A. SORDAHL uptake by these mitochondria have revealed differences in the a p p a r e n t Km and Vm,x for t r a n s p o r t in mitochondria obtained from the species' hearts.
LV
Succ,
Succ,
[co*]~/M
MATERIALS AND METHODS Mitochondria were isolated by previously described techniques (Sordahl et al., 1971) from the right and left ventricular tissue of Canisfamiliaris (dog), Fells catus (cat), Oryctolagus cuniculus (rabbit) and Sus scrofa (pig). Essentially this involved the isolation of mitochondria in a medium consisting of: 0.18 M KCI, 10mM EGTA, and 0.5% bovine serum albumin (pH 7.2). The initial mitochondrial pellet was subsequently suspended in a medium containing 1 mM EGTA and recentrifuged. In the second "wash" procedure the concentration of EGTA was reduced to 0.1 mM. The final suspension of mitochondria was in the above isolation medium minus EGTA. Mitochondrial respiratory activity was measured by polarographic means (Sordahl et al., i971). Cytochrome oxidase activity (mitochondrial marker enzyme) was determined in the various homogenate fractions as well as the mitochondrial fractions by an established spectrophotometric technique (Rieske, 1967). The initial velocities of calcium uptake were determined using the chelometric dye murexide (ammonium purpurate) in a DW-2 dual-wavelength spectrophotometer (American Instrument Company) at the wavelength pair 541 507nm by a modification of previously described methods (Sordahl et al., 1971; Scarpa, 1972). The reaction medium for calcium uptake consisted of: 0.25 M sucrose, 5 mM Tris-HC1 (pH 7.2), 70 mM NaCI or KCI, 8 mM inorganic phosphate, 50 #M murexide, 5 ,ug rotenone and 3 mg mitochondrial protein in a total vol of 3.0ml. Succinate was added at a final concentration of 5 mM and the temperature of the reaction was 30°C. Mitochondrial protein was determined by a Biuret method Jacobs et al., 1956). RESULTS Figure 1 are representative oxygen electrode tracings obtained from m i t o c h o n d r i a isolated from the right a n d left ventricles of Canis, Fells, S. scrofi~ a n d O. cuniculus heart. It can be seen that the efficiency LEFT V
oRabbit
Do9
RV
[co"l~M O-
, I rain ,
Fig. 2. Comparativedual-beam spectrophotometric tracings of calcium uptake (downward deflection) by heart mitochondria from left (LV) and right (RV) ventricles of rabbit and dog. Addition of the respiratory substrate succinate (Succ.) produces a rapid linear uptake of calcium by the mitochondrial preparations. The numbers to the right of each trace are the rates of calcium uptake expressed in nmoles of calcium taken up/min per mg mitochondrial protein. See "Methods" for further details. of oxidative phosphorylation ( A D P : O ) is the same in all of the preparations, while a consistent 20-25% lower rate of phosphorylating respiratory activity (State 3, QO2) is observed in the right ventricular mitochondrial preparations c o m p a r e d to those obtained from the left ventricular area. Although the absolute values for the rates of respiration (State 3, QO2) varied in each of the mitochondrial heart preparations from day to day, in each individual heart the comparison between right a n d left ventricular mitochondrial preparations was always significantly different. Figure 2 are representative dual-beam spectrop h o t o m e t r i c tracings of the respiration-supported calcium uptake by mitochondria extracted from the right A£)P
ADP i =3.0
A~
27
B~
C~
D__
151 ~
133 ~
124
RIGHT V
IO01nAO2 I
rain.
Fig. 1. Comparative oxygen electrode tracings of heart mitochondria isolated from the left (upper traces) and right (lower traces) ventricles of four species. A. Rabbit. B. Pig. C. Dog. D. Cat. Addition of ADP (550nmoles) produces rapid State 3 phosphorylating rates of respiration. The numbers to the left of each trace represent the rate of oxygen consumption in State 3 expressed in natoms of oxygen/min per mg mitochondrial protein. The A D P : O ratio is the efficiency of the consumption of oxygen to the phosphorylation of ADP to ATP. Substrate is glutamate-malate. See "Methods" for further details.
Mitochondrial functions from ventricular myocardium of four mammalian species and left ventricles of Canis and O. cuniculus heart. As can be seen the right ventricular mitochondria exhibit rates of calcium uptake lower than those of the left ventricular mitochondria from the same species' heart. These data are consistent with the observed differences in respiratory activities (Fig. 1). Similar results were obtained with comparative right and left ventricular mitochondrial preparations from the S. scrofa and Felis heart. Figure 3 are the curves obtained from the left ventricular mitochondria of the four species for maximal rates of calcium uptake with increasing concentrations of calcium. It can be seen that S. scrofa and 0. cuniculus heart mitochondria exhibit an apparent K,, of approx 50-60 #M calcium and a Vm,xat approx 200 #M with rates of calcium uptake at this point of approx 250 nmoles/min per mg mitochondrial protein. Mitochondria from the left ventricle of Canis have a somewhat lower Km (30-40 #M) and Vmaxat approx 150 #M calcium. In contrast, Felis heart mitochondria exhibit a markedly higher Km value (~ 100#M) and reach a Vmaxmuch sooner with the actual rates of calcium uptake considerably lower than those of the other three species studied. Similar values were obtained with right ventricular mitochondria from all four species (data not shown). Since the possibility that significantly different amounts of active mitochondrial protein may have been obtained from the right and left ventricles, cytochrome oxidase activity was measured in all total homogenates as well as the respective mitochondrial and other subcellular fractions. Figure 4 shows that essentially the same relative amounts of active mitochondrial protein (cytochrome oxidase) were obtained from the right and left ventricles of Canis and O. cuniculus with respect to per cent activity of the total homogenate. Similar values were obtained for right and left ventricular mitochondrial preparations from Felis and S. scrofa (data not shown). DISCUSSION
The data show that respiratory activity of mitochondria isolated from right ventricular myocardium is 20-25~o lower than that of mitochondria isolated from the left ventricular myocardium of the same heart. This finding was consistent in all four species of mammalian hearts studied. The rates of respiration 500 ~
x x
x
s
~
~ ,
•
"
__o. ~
E
o
E
0
~ 0
F 25
E
I 75
I
I 125
~-
-T ....... i 175
] 225
Fig. 3. Initial rates of calcium uptake by left ventricular mitochondrial preparations at varying concentrations of calcium. Pig ( H ) , rabbit (x x ), dog ( ~ O), and cat (V V).
341
I00 -
75% Total Cytochrome
Oxidase Activity
50-
25i
Rabbit
Dog
Fig. 4. The per cent yield of cytochrome oxidase activity of the left (LV) and right (RV) ventricular mitochondrial preparations from rabbit and dog hearts. The bars indicate the per cent cytochrome oxidase activity recovered in the mitochondrial fraction compared to the cytochrome oxidase activity of the total initial homogenates. (100~o). during active phosphorylation of ADP (QO2; State 3; Fig. 1) can be considered to be a quantitative measure of the ability of mitochondria to produce ATP. That is, the State 3 rate of respiration represents the maximal oxidation of substrates in order to phosphorylate ADP to ATP. Respiratory-substrate supported uptake of calcium by mitochondria from right ventricular myocardium was also less than that found in comparable mitochondrial preparations from left ventricle (Fig. 2). This indicates that an energy-linked function of mitochondria, such as active cation transport, is less in right ventricular mitochondria compared to those from the left ventricle (Fig. 2). Based on morphometric studies showing a 20~o lower volumetric density of mitochondria in right ventricular myocardium compared to left, Laguens (1971) suggested that the lower contractile activity of right ventricle should have a lower energy demand (i.e. mitochondria). This explanation may be applicable to the data reported here. In a recent report, Wikman-Coffelt et al. (1975) found the myosin ATPase activities of canine right ventricle to be approx 20~o lower than that of left ventricular preparations, further suggesting lesser energy demands in right myocardium compared to left. However, if fewer mitochondria exist per unit volume in right ventricular myocardium (Laguens, 1971), it does not necessarily hold that their "functional capacity should also be reduced because of lesser energy demands in the right myocardium. Although, at present, no other explanation seems apparent. Preliminary evidence in this laboratory indicates that possible differences in endogenous mitochondrial magnesium content may be affecting the kinetics of calcium transport in the various preparations that were studied. Exogenous magnesium has been found to have a marked effect on initial rates of mitochondrial calcium uptake and phosphorylation capacity (Sordahl, 1975). Additional comparative studies of cytochrome content and H ÷ ejection in right and left ventricular mitochondria are currently in progress. It should also be noted, that consistent differences in mitochondrial functions from right and left ventricular myocardium make comparative pathologic studies invalid when one side of the heart serves as a control to the other. The physiological significance of mitochondrial calcium transport in heart remains speculative (Ktibler & Shinebourne, 1971; Schwartz et al., 1973). The
342
Louls A. SORDAHL
results reported here for initial velocities of calcium uptake (Fig. 3) are similar to those obtained by Scarpa & Graziotti (1973) in m a m m a l i a n heart mitochondria. The data in Fig. 3 also indicate m a r k e d differences a m o n g species with respect to a p p a r e n t Km and Vrnax for mitochondrial calcium transport. This is particularly true of mitochondria isolated from Felis myocardium. It has been suggested that mitoc h o n d r i a may play a significant role in the maintenance of ionic (calcium) homeostasis within the cell (Bygrave, 1967; Kiibler & Shinebourne, 1971; Borle, 1973). If this is true, the data in Fig. 3 would indicate that either the calcium metabolism between various species of m a m m a l i a n hearts is quite different or that the role of m i t o c h o n d r i a in maintaining calcium ion homeostasis is considerably different between various species' hearts. The m a r k e d differences in calcium transport capability of Felis heart m i t o c h o n d r i a compared to the other m a m m a l i a n species studied may have significant bearing on the function of this organelle system in the cardiac physiology of Felis heart. This is notable in light of the fact that a n u m b e r of animal models of cardiac hypertrophy and failure have utilized Fells heart and in particular, the Felis papillary muscle preparation for studies of cardiac pathology. SUMMARY M i t o c h o n d r i a isolated from the right ventricular myocardium of four m a m m a l i a n species exhibited 20-25% lower respiratory activity c o m p a r e d to mitoc h o n d r i a from the left ventricular myocardium of the same heart. Respiration-supported active accumulation of calcium by right ventricular mitochondrial preparations was similarly lower in activity compared to left ventricular mitochondrial preparations. In addition, m a r k e d differences in the a p p a r e n t K,, and Vmax values for active calcium transport in the four m a m m a l i a n heart species were found. The differences between right a n d left ventricular mitochondrial functions may be ascribed to the lower energy demands of the right ventricle as c o m p a r e d to the left in mammalian heart. The differences in the calcium uptake functions of m i t o c h o n d r i a extracted from the different species of m a m m a l i a n heart may be related to the cardiac physiology a n d the role of m i t o c h o n d r i a in maintaining calcium ion homeostasis within these cells. However, the significance of these differences at present remains unclear. Acknowledgements--The assistance of Michael L. Stewart in these studies is greatly appreciated. REFERENCES
ALPERT N. R. (1971) Cardiac Hypertrophy. Academic Press, New York. ANVERSA P., VITALI-MAZZA L., VISIOLI 0. & MARCHETT1 G. (1971 ) Experimental cardiac hypertrophy: a quantitative ultra-structural study in the compensatory stage. J. molec. Cell. Cardiol. 3. 213 227. BAJUSZ E. & RONA G. (1972) Myocardiology, Vol. 1 University Park Press, Baltimore. BISHOP S. P. & COLE C. R. (1969) Ultrastructural changes in the canine myocardium with right ventricular hypertrophy and congestive heart failure. Lab. Invest. 20. 219-229. BORLE A. B. (1973) Calcium metabolism at the cellular level. Fedn Proc. Fedn Am. Socs exp. Biol. 32, 1944 1950.
BRAIMBRIDGE M. V., DARRACOTT S., CHAYEN J. & BITENSKY L. (1973) A cellular chemical comparison of left ventricular wall myocardium and papillary muscle in man. J. thorac. Cardiovasc. Sury. 65, 722-726. BYGRAVE F. L. (1967) The ionic environment and metabolic control. Nature, Lond. 214, 667-671. CARAFOLI E. & LEHN1NGER A. L. (1971) A survey of the interaction of calcium ions with mitochondria from different tissues and species. Biochem. J. 122, 681-690. GOLDSTEIN M. A., SORDAHL L. A. t~ SCHWARTZ A. (1974) Ultrastructural analysis of left ventricular hypertrophy in rabbits. J. molec. Cell. Cardiol. 6, 265-274. HORT W. (1971) Quantitative morphology and structural dynamics of the myocardium. In Methods and Achievements in Experimental Pathology (Edited by BaJusz E. 8/, JASMIN G.), go|. 5, pp. 3-21. S. Karger Press, Basel. JACOBS E. E., JACOB M., SANADI D. R. & BRADLEY L. B. (1956) Uncoupling of oxidative phosphorylation by cadmium ion. J. biol. Chem. 223, 147 156. Ki)BLER W. & SHINEBOURNE E. A. (1971) Calcium and the mitochondria. In Calcium and the Heart (Edited by HARRIS P. & OPlE L. H.), pp. 93-123. Academic Press, New York. LAGUENS R. (1971) Morphometric study of myocardial mitochondria in the rat. J. Cell Biol. 48, 673-676. LINDENMAYER G. E., SORDAHL L. A., HARIGAYA S., ALLEN J. C., BESCH H. R. JR. & SCHWARTZ A. (1971) Some biochemical studies on subcellular systems isolated from fresh recipient human cardiac tissue obtained during transplantation. Am. J. Cardiol. 27. 277-283. MEERSON F. Z., ZALETAYEVAT. A., LAGUTCHEV S. S. & PSHENNIKOVA M. G. (1964) Structure and mass of mitochondria in the process of compensatory hyperfunction and hypertrophy of the heart. Expl Cell Res. 36, 568-578. PAGE E. • MCCALLISTERL. P. (1973) Quantitative electron microscopic description of heart muscle cells. Application to normal, hypertrophied and thyroxin-stimulated hearts. Am. J. Cardiol. 31, 172-181. RIESKE J. S. (1967) The quantitative determination of mitochondrial hemoproteins. In Methods in Enzymology (Edited by ESTABROOK R. W. ~; PULLMAN M. E.), Vol. 10, pp. 488-493. Academic Press, New York. SCARPA A. (1972) Spectrophotometric measurement of calcium by murexide. In Methods in Enzymology (Edited by SAN P1ETROA.), Vol. 24, pp. 343 351. Academic Press, New York. SCARPA A. & GRAZIOTTI P. (1973) Mechanisms for intracellular calcium regulation in heart I. Stopped-flow measurements of Ca z ÷ uptake by cardiac mitochondria. J. gen. Physiol. 62, 756-772. SCHWARTZ A., SORDAHL L. A., ENTMAN M. L., ALLEN J. C., REDDY Y. S., GOLDSTEIN M. A., LUCHI R. J. & WYBORNY L. W. (1973) Abnormal biochemistry in myocardial failure. Am. J. Cardiol. 32. 407 422. SORDAHL L. A. (1975) Effects of magnesium, ruthenium red and the antibiotic ionophore A-23187 on initial rates of calcium uptake and release by heart mitochondria. Archs Biochem. Biophys. 167. 104-115. SORDAHL L. A., BESCH H. R., ALLEN J. C., CROW C., LINDENMAYER G. E. & SCHWARTZ A. (1971) Enzymatic aspects of the cardiac muscle cell: Mitochondria, sarcoplasmic reticulum and monovalent cation active transport system. In Methods and Achievements in Experimental Pathology (Edited by BAJUSZ E. & JASMIN G.). Vol. 5. pp. 287 346. S. Karger Press, Basel. W1KMAN-COFFELT J., FENNER C., SMITH A. & MASON D. T. (1975) Comparative analysis of the kinetics and subunits of myosins from canine skeletal muscle and cardiac tissue. J. biol. Chem. 250. 1257-1262. WOLLENBERGER A. & SCHULZE W. (1961) Mitochondrial alterations in the myocardium of dogs with ~/ortic stenosis. J. biophys, biochem. Cytol. 10, 285-288.
DIFFERENCES IN MITOCHONDRIAL F U N C T I O N S FROM RIGHT A N D LEFT VENTRICULAR M Y O C A R D I U M OF FOUR MAMMALIAN SPECIES* LOUIS A. SORDAHL Division of Biochemistry, University of Texas Medical Branch, Galveston, TX 77550, U.S.A. (Received 22 July 1975)
Abstract--1. Mitochondria were isolated from the right and left ventricles of Canis familiaris, Felis catus, Oryctolagus cuniculus and Sus scrofa hearts. 2. A consistent 20-25~o lower rate of phosphorylating respiration (State 3) was found in right ventricular mitochondria compared to left in all four species. 3. Measurement of initial velocities of calcium uptake using dual-beam spectrophotometry revealed marked differences in the apparent KM and Vmaxvalues between the mitochondrial preparations from the four species. 4. The results indicate significant differences in mitochondrial respiratory activity between right and left myocardium and further suggest possible differences in the role of mitochondria in maintaining cellular calcium homeostasis in the various species' hearts.
INTRODUCTION
independently determined by Page & McCallister (1973) for normal rat left ventricle. Carafoli & Lehninger (1971) have surveyed mitochondrial calcium transport in a number of vertebrate and invertebrate tissues, including heart. These investigators (Carafoli & Lehninger, 1971) found no significant difference in mitochondrial calcium transport functions in all mammalian tissues studied. Recently, Braimbridge et al. (1973) demonstrated differences in enzyme levels of left ventricular free wall and papillary muscles in patients undergoing open-heart surgery. Several years ago this investigator observed a consistent difference in mitochondrial functions in right and left ventricular tissue obtained from cardiac transplant recipients (Lindenmayer et al., 1971). Although these hearts were in an abnormal state and a good deal of drug intervention preceded obtaining the tissues for subsequent biochemical analyses, this observation pointed out the possible differences in the functionality of mitochondria from various parts of the ventricular myocardium. Mitochondria not only constitute a significant portion of the total cytoplasmic volume of the myocardial cell, but under normal circumstances are the sole source of ATP for cellular processes. Energy production in the heart cell is directly related to the oxidative processes of mitochondria. Further, this organelle system of the heart may very well be involved in the movements of calcium related to excitation-contraction coupling in the myocardial cell. Evidence to date does not establish a direct role for mitochondria in the calcium fluxes related to myocardial contractility, but the possibility that mitochondria may play a role in maintaining the ionic homeostasis of the heart cell has been suggested by several investigators (Bygrave, 1967; Kiibler & Shinebourne, 1971 ; Borle, 1973). This report describes distinct and consistent differences in *Supported by grants from USPHS, 5 respiratory activity of mitochondria isolated from the SO1-RR-05427-12, HL 14828, USAF, AFOSR 74-2622 and right and left ventricular myocardium of four mammalian species. Determination of initial velocities of Texas Heart Association. 339
It is well known that the mammalian heart, at birth, is relatively equal in size with respect to the number of fibers in the right and left myocardium (Hort, 1971). During early post-natal development the left heart (ventricle) undergoes considerable enlargement relative to the right, yet the number of fibers on both sides remains constant (Hort, 1971). A number of studies have been reported (Alpert, 1971; Bajusz & Rona, 1972) on functional and structural changes of mammalian heart in experimentally induced cardiac pathologies. Specific changes in mitochondrial numbers, mass, volume and distribution have been shown in both right and left ventricular hypertrophy (Wollenberger & Schulze, 1961 ; Meerson, et al., 1964; Bishop & Cole, 1969). Using quantitative biometric techniques Anversa et aL (1971), Page & McCallister (1973); Goldstein et al. (1974) have demonstrated significant decreases in the mitochondria-myofibril ratio during left ventricular hypertrophy induced by aortic constriction. The above studies indicate marked ultrastructural changes can occur in ventricular myocardium under pathologic circumstances. Alterations in mitochondrial functions during cardiac hypertrophy and failure have also been observed (Sordahl et aL, 1971). However, very little has been reported on comparative ultrastructur¢ and function of mitochondria in normal myocardium. Laguens (1971) has reported 20~o fewer mitochondria in the right ventricle of normal rat myocardium compared to left. Employing quantitative hiometric techniques, Laguens (1971) found 36.6~o of the cytoplasmic volume occupied by mitochondria in the left ventricle and 29.3?/0 in the right. This value for left ventricular mitochondrial volumetric density is in close agreement to that of 35.8~o
340 calcium marked calcium various
Louis A. SORDAHL uptake by these mitochondria have revealed differences in the a p p a r e n t Km and Vm,x for t r a n s p o r t in mitochondria obtained from the species' hearts.
LV
Succ,
Succ,
[co*]~/M
MATERIALS AND METHODS Mitochondria were isolated by previously described techniques (Sordahl et al., 1971) from the right and left ventricular tissue of Canisfamiliaris (dog), Fells catus (cat), Oryctolagus cuniculus (rabbit) and Sus scrofa (pig). Essentially this involved the isolation of mitochondria in a medium consisting of: 0.18 M KCI, 10mM EGTA, and 0.5% bovine serum albumin (pH 7.2). The initial mitochondrial pellet was subsequently suspended in a medium containing 1 mM EGTA and recentrifuged. In the second "wash" procedure the concentration of EGTA was reduced to 0.1 mM. The final suspension of mitochondria was in the above isolation medium minus EGTA. Mitochondrial respiratory activity was measured by polarographic means (Sordahl et al., i971). Cytochrome oxidase activity (mitochondrial marker enzyme) was determined in the various homogenate fractions as well as the mitochondrial fractions by an established spectrophotometric technique (Rieske, 1967). The initial velocities of calcium uptake were determined using the chelometric dye murexide (ammonium purpurate) in a DW-2 dual-wavelength spectrophotometer (American Instrument Company) at the wavelength pair 541 507nm by a modification of previously described methods (Sordahl et al., 1971; Scarpa, 1972). The reaction medium for calcium uptake consisted of: 0.25 M sucrose, 5 mM Tris-HC1 (pH 7.2), 70 mM NaCI or KCI, 8 mM inorganic phosphate, 50 #M murexide, 5 ,ug rotenone and 3 mg mitochondrial protein in a total vol of 3.0ml. Succinate was added at a final concentration of 5 mM and the temperature of the reaction was 30°C. Mitochondrial protein was determined by a Biuret method Jacobs et al., 1956). RESULTS Figure 1 are representative oxygen electrode tracings obtained from m i t o c h o n d r i a isolated from the right a n d left ventricles of Canis, Fells, S. scrofi~ a n d O. cuniculus heart. It can be seen that the efficiency LEFT V
oRabbit
Do9
RV
[co"l~M O-
, I rain ,
Fig. 2. Comparativedual-beam spectrophotometric tracings of calcium uptake (downward deflection) by heart mitochondria from left (LV) and right (RV) ventricles of rabbit and dog. Addition of the respiratory substrate succinate (Succ.) produces a rapid linear uptake of calcium by the mitochondrial preparations. The numbers to the right of each trace are the rates of calcium uptake expressed in nmoles of calcium taken up/min per mg mitochondrial protein. See "Methods" for further details. of oxidative phosphorylation ( A D P : O ) is the same in all of the preparations, while a consistent 20-25% lower rate of phosphorylating respiratory activity (State 3, QO2) is observed in the right ventricular mitochondrial preparations c o m p a r e d to those obtained from the left ventricular area. Although the absolute values for the rates of respiration (State 3, QO2) varied in each of the mitochondrial heart preparations from day to day, in each individual heart the comparison between right a n d left ventricular mitochondrial preparations was always significantly different. Figure 2 are representative dual-beam spectrop h o t o m e t r i c tracings of the respiration-supported calcium uptake by mitochondria extracted from the right A£)P
ADP i =3.0
A~
27
B~
C~
D__
151 ~
133 ~
124
RIGHT V
IO01nAO2 I
rain.
Fig. 1. Comparative oxygen electrode tracings of heart mitochondria isolated from the left (upper traces) and right (lower traces) ventricles of four species. A. Rabbit. B. Pig. C. Dog. D. Cat. Addition of ADP (550nmoles) produces rapid State 3 phosphorylating rates of respiration. The numbers to the left of each trace represent the rate of oxygen consumption in State 3 expressed in natoms of oxygen/min per mg mitochondrial protein. The A D P : O ratio is the efficiency of the consumption of oxygen to the phosphorylation of ADP to ATP. Substrate is glutamate-malate. See "Methods" for further details.
Mitochondrial functions from ventricular myocardium of four mammalian species and left ventricles of Canis and O. cuniculus heart. As can be seen the right ventricular mitochondria exhibit rates of calcium uptake lower than those of the left ventricular mitochondria from the same species' heart. These data are consistent with the observed differences in respiratory activities (Fig. 1). Similar results were obtained with comparative right and left ventricular mitochondrial preparations from the S. scrofa and Felis heart. Figure 3 are the curves obtained from the left ventricular mitochondria of the four species for maximal rates of calcium uptake with increasing concentrations of calcium. It can be seen that S. scrofa and 0. cuniculus heart mitochondria exhibit an apparent K,, of approx 50-60 #M calcium and a Vm,xat approx 200 #M with rates of calcium uptake at this point of approx 250 nmoles/min per mg mitochondrial protein. Mitochondria from the left ventricle of Canis have a somewhat lower Km (30-40 #M) and Vmaxat approx 150 #M calcium. In contrast, Felis heart mitochondria exhibit a markedly higher Km value (~ 100#M) and reach a Vmaxmuch sooner with the actual rates of calcium uptake considerably lower than those of the other three species studied. Similar values were obtained with right ventricular mitochondria from all four species (data not shown). Since the possibility that significantly different amounts of active mitochondrial protein may have been obtained from the right and left ventricles, cytochrome oxidase activity was measured in all total homogenates as well as the respective mitochondrial and other subcellular fractions. Figure 4 shows that essentially the same relative amounts of active mitochondrial protein (cytochrome oxidase) were obtained from the right and left ventricles of Canis and O. cuniculus with respect to per cent activity of the total homogenate. Similar values were obtained for right and left ventricular mitochondrial preparations from Felis and S. scrofa (data not shown). DISCUSSION
The data show that respiratory activity of mitochondria isolated from right ventricular myocardium is 20-25~o lower than that of mitochondria isolated from the left ventricular myocardium of the same heart. This finding was consistent in all four species of mammalian hearts studied. The rates of respiration 500 ~
x x
x
s
~
~ ,
•
"
__o. ~
E
o
E
0
~ 0
F 25
E
I 75
I
I 125
~-
-T ....... i 175
] 225
Fig. 3. Initial rates of calcium uptake by left ventricular mitochondrial preparations at varying concentrations of calcium. Pig ( H ) , rabbit (x x ), dog ( ~ O), and cat (V V).
341
I00 -
75% Total Cytochrome
Oxidase Activity
50-
25i
Rabbit
Dog
Fig. 4. The per cent yield of cytochrome oxidase activity of the left (LV) and right (RV) ventricular mitochondrial preparations from rabbit and dog hearts. The bars indicate the per cent cytochrome oxidase activity recovered in the mitochondrial fraction compared to the cytochrome oxidase activity of the total initial homogenates. (100~o). during active phosphorylation of ADP (QO2; State 3; Fig. 1) can be considered to be a quantitative measure of the ability of mitochondria to produce ATP. That is, the State 3 rate of respiration represents the maximal oxidation of substrates in order to phosphorylate ADP to ATP. Respiratory-substrate supported uptake of calcium by mitochondria from right ventricular myocardium was also less than that found in comparable mitochondrial preparations from left ventricle (Fig. 2). This indicates that an energy-linked function of mitochondria, such as active cation transport, is less in right ventricular mitochondria compared to those from the left ventricle (Fig. 2). Based on morphometric studies showing a 20~o lower volumetric density of mitochondria in right ventricular myocardium compared to left, Laguens (1971) suggested that the lower contractile activity of right ventricle should have a lower energy demand (i.e. mitochondria). This explanation may be applicable to the data reported here. In a recent report, Wikman-Coffelt et al. (1975) found the myosin ATPase activities of canine right ventricle to be approx 20~o lower than that of left ventricular preparations, further suggesting lesser energy demands in right myocardium compared to left. However, if fewer mitochondria exist per unit volume in right ventricular myocardium (Laguens, 1971), it does not necessarily hold that their "functional capacity should also be reduced because of lesser energy demands in the right myocardium. Although, at present, no other explanation seems apparent. Preliminary evidence in this laboratory indicates that possible differences in endogenous mitochondrial magnesium content may be affecting the kinetics of calcium transport in the various preparations that were studied. Exogenous magnesium has been found to have a marked effect on initial rates of mitochondrial calcium uptake and phosphorylation capacity (Sordahl, 1975). Additional comparative studies of cytochrome content and H ÷ ejection in right and left ventricular mitochondria are currently in progress. It should also be noted, that consistent differences in mitochondrial functions from right and left ventricular myocardium make comparative pathologic studies invalid when one side of the heart serves as a control to the other. The physiological significance of mitochondrial calcium transport in heart remains speculative (Ktibler & Shinebourne, 1971; Schwartz et al., 1973). The
342
Louls A. SORDAHL
results reported here for initial velocities of calcium uptake (Fig. 3) are similar to those obtained by Scarpa & Graziotti (1973) in m a m m a l i a n heart mitochondria. The data in Fig. 3 also indicate m a r k e d differences a m o n g species with respect to a p p a r e n t Km and Vrnax for mitochondrial calcium transport. This is particularly true of mitochondria isolated from Felis myocardium. It has been suggested that mitoc h o n d r i a may play a significant role in the maintenance of ionic (calcium) homeostasis within the cell (Bygrave, 1967; Kiibler & Shinebourne, 1971; Borle, 1973). If this is true, the data in Fig. 3 would indicate that either the calcium metabolism between various species of m a m m a l i a n hearts is quite different or that the role of m i t o c h o n d r i a in maintaining calcium ion homeostasis is considerably different between various species' hearts. The m a r k e d differences in calcium transport capability of Felis heart m i t o c h o n d r i a compared to the other m a m m a l i a n species studied may have significant bearing on the function of this organelle system in the cardiac physiology of Felis heart. This is notable in light of the fact that a n u m b e r of animal models of cardiac hypertrophy and failure have utilized Fells heart and in particular, the Felis papillary muscle preparation for studies of cardiac pathology. SUMMARY M i t o c h o n d r i a isolated from the right ventricular myocardium of four m a m m a l i a n species exhibited 20-25% lower respiratory activity c o m p a r e d to mitoc h o n d r i a from the left ventricular myocardium of the same heart. Respiration-supported active accumulation of calcium by right ventricular mitochondrial preparations was similarly lower in activity compared to left ventricular mitochondrial preparations. In addition, m a r k e d differences in the a p p a r e n t K,, and Vmax values for active calcium transport in the four m a m m a l i a n heart species were found. The differences between right a n d left ventricular mitochondrial functions may be ascribed to the lower energy demands of the right ventricle as c o m p a r e d to the left in mammalian heart. The differences in the calcium uptake functions of m i t o c h o n d r i a extracted from the different species of m a m m a l i a n heart may be related to the cardiac physiology a n d the role of m i t o c h o n d r i a in maintaining calcium ion homeostasis within these cells. However, the significance of these differences at present remains unclear. Acknowledgements--The assistance of Michael L. Stewart in these studies is greatly appreciated. REFERENCES
ALPERT N. R. (1971) Cardiac Hypertrophy. Academic Press, New York. ANVERSA P., VITALI-MAZZA L., VISIOLI 0. & MARCHETT1 G. (1971 ) Experimental cardiac hypertrophy: a quantitative ultra-structural study in the compensatory stage. J. molec. Cell. Cardiol. 3. 213 227. BAJUSZ E. & RONA G. (1972) Myocardiology, Vol. 1 University Park Press, Baltimore. BISHOP S. P. & COLE C. R. (1969) Ultrastructural changes in the canine myocardium with right ventricular hypertrophy and congestive heart failure. Lab. Invest. 20. 219-229. BORLE A. B. (1973) Calcium metabolism at the cellular level. Fedn Proc. Fedn Am. Socs exp. Biol. 32, 1944 1950.
BRAIMBRIDGE M. V., DARRACOTT S., CHAYEN J. & BITENSKY L. (1973) A cellular chemical comparison of left ventricular wall myocardium and papillary muscle in man. J. thorac. Cardiovasc. Sury. 65, 722-726. BYGRAVE F. L. (1967) The ionic environment and metabolic control. Nature, Lond. 214, 667-671. CARAFOLI E. & LEHN1NGER A. L. (1971) A survey of the interaction of calcium ions with mitochondria from different tissues and species. Biochem. J. 122, 681-690. GOLDSTEIN M. A., SORDAHL L. A. t~ SCHWARTZ A. (1974) Ultrastructural analysis of left ventricular hypertrophy in rabbits. J. molec. Cell. Cardiol. 6, 265-274. HORT W. (1971) Quantitative morphology and structural dynamics of the myocardium. In Methods and Achievements in Experimental Pathology (Edited by BaJusz E. 8/, JASMIN G.), go|. 5, pp. 3-21. S. Karger Press, Basel. JACOBS E. E., JACOB M., SANADI D. R. & BRADLEY L. B. (1956) Uncoupling of oxidative phosphorylation by cadmium ion. J. biol. Chem. 223, 147 156. Ki)BLER W. & SHINEBOURNE E. A. (1971) Calcium and the mitochondria. In Calcium and the Heart (Edited by HARRIS P. & OPlE L. H.), pp. 93-123. Academic Press, New York. LAGUENS R. (1971) Morphometric study of myocardial mitochondria in the rat. J. Cell Biol. 48, 673-676. LINDENMAYER G. E., SORDAHL L. A., HARIGAYA S., ALLEN J. C., BESCH H. R. JR. & SCHWARTZ A. (1971) Some biochemical studies on subcellular systems isolated from fresh recipient human cardiac tissue obtained during transplantation. Am. J. Cardiol. 27. 277-283. MEERSON F. Z., ZALETAYEVAT. A., LAGUTCHEV S. S. & PSHENNIKOVA M. G. (1964) Structure and mass of mitochondria in the process of compensatory hyperfunction and hypertrophy of the heart. Expl Cell Res. 36, 568-578. PAGE E. • MCCALLISTERL. P. (1973) Quantitative electron microscopic description of heart muscle cells. Application to normal, hypertrophied and thyroxin-stimulated hearts. Am. J. Cardiol. 31, 172-181. RIESKE J. S. (1967) The quantitative determination of mitochondrial hemoproteins. In Methods in Enzymology (Edited by ESTABROOK R. W. ~; PULLMAN M. E.), Vol. 10, pp. 488-493. Academic Press, New York. SCARPA A. (1972) Spectrophotometric measurement of calcium by murexide. In Methods in Enzymology (Edited by SAN P1ETROA.), Vol. 24, pp. 343 351. Academic Press, New York. SCARPA A. & GRAZIOTTI P. (1973) Mechanisms for intracellular calcium regulation in heart I. Stopped-flow measurements of Ca z ÷ uptake by cardiac mitochondria. J. gen. Physiol. 62, 756-772. SCHWARTZ A., SORDAHL L. A., ENTMAN M. L., ALLEN J. C., REDDY Y. S., GOLDSTEIN M. A., LUCHI R. J. & WYBORNY L. W. (1973) Abnormal biochemistry in myocardial failure. Am. J. Cardiol. 32. 407 422. SORDAHL L. A. (1975) Effects of magnesium, ruthenium red and the antibiotic ionophore A-23187 on initial rates of calcium uptake and release by heart mitochondria. Archs Biochem. Biophys. 167. 104-115. SORDAHL L. A., BESCH H. R., ALLEN J. C., CROW C., LINDENMAYER G. E. & SCHWARTZ A. (1971) Enzymatic aspects of the cardiac muscle cell: Mitochondria, sarcoplasmic reticulum and monovalent cation active transport system. In Methods and Achievements in Experimental Pathology (Edited by BAJUSZ E. & JASMIN G.). Vol. 5. pp. 287 346. S. Karger Press, Basel. W1KMAN-COFFELT J., FENNER C., SMITH A. & MASON D. T. (1975) Comparative analysis of the kinetics and subunits of myosins from canine skeletal muscle and cardiac tissue. J. biol. Chem. 250. 1257-1262. WOLLENBERGER A. & SCHULZE W. (1961) Mitochondrial alterations in the myocardium of dogs with ~/ortic stenosis. J. biophys, biochem. Cytol. 10, 285-288.
