Pfl/igers Arch. 358, 195--201 (1975) 9 by Springer-Verlag 1975

The Comparative Effects of [Ca2§ and [Mg2§ on Tension Generation in the Fibers of Skinned Frog Skeletal Muscle and Mechanically Disrupted Rat Ventricular Cardiac Muscle W . G l e n n L. K e r r i e k a n d Sue K . B o l i t h o D o n a l d s o n Departments of Physiology and Biophysics and Physiological Nursing, University of Washington School of Medicine, Seattle, Washington, U.S.A. Received April 14, 1975

Summary. The effects of intracellular Mg2+ on Ca~+-activated isometric tension generation in rat cardiac muscle fibers and frog skeletal muscle were compared. The membranous sarcolemmal barrier was removed from rat cardiac muscle fibers by mechanical disruption and from frog skeletal muscle by skinning. Tension was recorded in the fibers in bathing soIutions of different Ca~+ eoneentratious and either 5 X 10-5 NI or 1 • 10-~ M Nfg~+ concentration (the same concentrations used in a previous study on single skinned frog skeletal muscle fibers [3]). The amount of Ca2+ required to activate the muscle increased with Mg2+ concentration for both rat ventrieular muscle and frog skeletal muscle. These data indicate that intracellular Mg~+ concentration could strongly modulate Ca2+-aetivated tension in cardiac muscle and that very similar molecular mechanisms are responsible for Ca2+-activated tension in rat ventricular muscle and frog skeletal muscle. The possible sites of action of Mg2+ on Ca2+-activated tension are discussed. Key words: Ca2+-Activated Isometric Tension -- Removal of Sarcolemma -Intracellular Ca2+ -- Intracellular Mg~+ -- Ca2+ Sensitivity. It has been established that the contraction of mammalian skeletal a n d c a r d i a c muscle is d e p e n d e n t on t h e b i n d i n g o f Ca *+ t o t r o p o n i n . T h e chemical p r o p e r t i e s o f t r o p o n i n in r e l a t i o n t o muscle c o n t r a c t i o n h a v e b e e n s t u d i e d e x t e n s i v e l y [20]. E b a s h i et al. [6] showed t h a t t r o p o n i n i s o l a t e d f r o m h e a r t muscle has a lesser affinity for Ca 2+ t h a n t r o p o n i n i s o l a t e d f r o m s k e l e t a l muscle. On t h e basis o f this i n f o r m a t i o n t h e relat i o n s h i p b e t w e e n Ca 2+ c o n c e n t r a t i o n a n d i s o m e t r i c t e n s i o n in c a r d i a c muscle s h o u l d differ from t h a t in s k e l e t a l muscle. This seems t o be confirmed b y t h e r e l a t i o n s h i p b e t w e e n Ca 2+ c o n c e n t r a t i o n a n d c e r t a i n biochemical m e a s u r e s o f c o n t r a c t i o n , such as s u p e r p r e c i p i t a t i o n o f a c t o m y o s i n gels a n d A T P a s e a c t i v i t y o f i s o l a t e d p r o t e i n s [6]. H o w e v e r , t h e c o m p a r a t i v e effects o f sarcoplasmic Ca 2+ c o n c e n t r a t i o n on i s o m e t r i c t e n s i o n g e n e r a t i o n in b o t h m e c h a n i c a l l y s k i n n e d (sarcolemma r e m o v e d ) c a r d i a c a n d skeletal muscle fibers h a v e n o t b e e n d e t e r m i n e d . Effects 14 PIlilgers Arch., Vol. 858

196

W. G. Kerrick and S. Donaldson

have b e e n established for o n l y m e c h a n i c a l l y s k i n n e d skeletal muscle [4, 8,16]. Mg ~+ c o n c e n t r a t i o n i n t h e physiological r a n g e (mM) [2,18] g r e a t l y influences t h e Ca 2+ s e n s i t i v i t y of t h e t e n s i o n - g e n e r a t i n g a p p a r a t u s of skeletal muscle [3,4,13], a n d t o t a l Mg c o n c e n t r a t i o n is t h o u g h to v a r y i n cardiac muscle [19]. Therefore, the c o m p a r a t i v e effects of Mg 2+ conc e n t r a t i o n o n Ca2+-activated t e n s i o n i n these two t y p e s of muscles m a y give clues to differences, if any, b e t w e e n their m e c h a n i s m s of control a n d m o d u l a t i o n of Ca2+-activated t e n s i o n generation. This p a p e r reports t h e r e l a t i o n s h i p b e t w e e n Ca ~+ a n d t e n s i o n i n m e c h a n i c a l l y s k i n n e d cardiac fibers a n d skeletal muscle fibers a n d t h e effect of Mg ~+ c o n c e n t r a t i o n o n this relationship.

Materials and Methods Preparation. Data were collected from small bundles of mechanically disrupted (sarcolemma broken)[7,12] rat ventricular tissue for comparison with data [3] previous collected under identical conditions from frog (Rana pipiens) skinned [16, 17] semitendinosns fibers. Thus, the muscles used were not only different in type (cardiac versus skeletal) but were also evolutionarily distinct (mammalian versus amphibian). Disrupted bundles of rat (Sprague-Dawley) cardiac fibers were prepared as described previously [12]. Skinning of the single frog fibers is also reported elsewhere [3]. Although the procedure for mechanical disruption of sarcolemma for cardiac muscle fibers differs from that used for the skeletal fibers, there appears to be no difference in data collected from skinned (peeled) versus homogenized skeletal fibers [14]. Tension Measurement. Cardiac fiber bundles or single skinned skeletal muscle fibers (approximately 1 mm long) were mounted by their ends in the forceps of a photodiode force transducer similar to that used by Hellam and Podolsky [3, 8,12]. The mounted cardiac fiber bundles and skeletal fibers were immersed sequentially in solutions of varying ionic composition and the isometric tension generated in each solution was continuously recorded. Baseline tension generation was established as the voltage output recorded with bundle immersed in a relaxing solution, described below. Only steady-state deviations in voltage from the baseline were used as measures of tension generation in each bathing solution. Using steady state tensions eliminated possible effects of sarcoplasmie reticulum or mitoehondria on the ionic environment of the contractile proteins as they would be manifested as tension transients. Bathing Solutions. Two sets of bathing solutions were used, each containing the amount of Ca2+ required for zero (relaxing solution with no added Ca~+) to maximum tension generation. The sets were identical in composition to two of the sets of bathing solutions used in a prior study of frog skeletal muscle fibers [3]. Mg2+ concentration was 5 • 10-5 M in the first and 1 • i0 -a 1K in the second set. The following concentrations were constant in all solutions: 1) 10-7 M H+, 2) 70 ml~ K +, 3) 7 mM total EGTA (ethyleneglycolbis-(#-amino-ethylether)-N,N,N',N'tetraacetic acid), 4) 2 mM MgATP2- (magnesium adenosine triphosphate), 5) 15 ~ CP2(creatine phosphate), and 6) 15 units/ec CPK (creatine phosphokinase). The major anion was C1-and ionic strength was maintained at 0.15 IV[ by varying imidazole concentration (pH -- 7.0). Room temperature was controlled at 20 4- 1~ C.

Tension in Disrupted Cardiac Cells

197

Protocol. The fiber bundles varied considerably in size, shape, and absolute maximum tension generation (kg/cm 2 cross-sectional area). The two protocols for data collection allowed for normalization of the data in order that data from different fiber bundles could be compared; they are described elsewhere [3]. Briefly, percentage of maximum tensions were obtained by dividing the force generated at a given submaximum Ca 2+ concentration and Mg ~+ concentration by the maximum force generated (at the same Yfg~+ concentration) by the same fiber or bundle immediately after the submaximum contraction. The ratio was multiplied by 100. Cardiac muscle fibers were discarded when maximum tension fell to less than 5 0 % of initial maximum value without a requirement as to a minimum initial tension as in our prior studies [3,13] because the bundles were very irregularly shaped and it was impossible to precisely determine the cross-sectional area. For the same reason, the finding that the absolute forces of the bundles were 1 0 - - 2 5 % less than those of skeletal muscle fibers of approximately the same diameter cannot be interpreted.

Results T h e r e l a t i o n s h i p s b e t w e e n Ca 2+ a n d M g 2+ c o n c e n t r a t i o n s for m e c h a n i c a l l y d i s r u p t e d c a r d i a c a n d s k i n n e d f r o g s k e l e t a l m u s c l e cells a r e s h o w n i n F i g . 1. I n o u r p r i o r s t u d y o f s k e l e t a l m u s c l e , w e d e t e r m i n e d t h a t t h e d a t a c o u l d n o t be d e s c r i b e d b y e q u a t i o n s for b i n d i n g o f Ca s+ IO0 9O 8O 7o 6o

~ 5o 0 0 5 x IO'sM

]

~2o 6,5

6.0

A,~

I x IO'~M

t

5.5

f

I

510 4.5 4.0 -lOgloCa~+(M)

3',~

~'.o

i

2,s

Fig. 1. Relationship between the percentages of maximum tension and -- logic[Ca2+] for cardiac and skeletal muscle at two concentrations of Mg 2+. The means of the data from skinned skeletal muscle fibers of the frog are plotted as open figures (o, ~) and those from mechanically disrupted ventricular fibers of the rat are plotted as closed figures (., A). Two different concentrations of free Mg=+: 5 • 10-5 (o, o) and 1 x 10-a Yl (~, ,) were employed. See text for ionic conditions. Sample size and ~ S.E.M. for each mean are shown in Table i. Constants for the smooth computer-fitted curves were obtained as described in the text. For the curve with [Mg2+] = 5 • 10-s M, n = 2.48 and Q = 10-1a.ss; and for the curve with [Nfg2+] = 1 • 10-3 ~ , n = 1.5 and Q = 10-~-~s 14"

198

W' G. Kerrick and S. Donaldson

Table 1. The mean and the standard errors of the means of the percentages of maximum tension and sample sizes of the indicated pCa, [Mg~+], and muscle fiber type a pCa

3.2 3.4 3.6 3.8 4.0 4.1 4.2 4.3 4.4 4.5 4.6 4.7 4.8 4.9 5.0 5.1 5.2 5.3 5.4 5.5 5.6 5.7 5.8 5.9 6.0

Muscle

Muscle

Cardiac Skeletal [Mg~+] = 5 • 10-5 M mean per cent maximum tension -4- standard error of the mean

Cardiac Skeletal [Mg2+] = 1 • 10-a M mean per cent maximum tension 4- standard error of the mean

1.00

1.00 1.00

96.0 (I)

1.00 95.7 4- 2.3 (6) 77.4 :J: 2.7 (8)

89.2 • 9.3 (5)

68.0 62.4 51.8 22.8

77.5 4- 9.4 (12) 66.4 4- 7.4 (11) 53.7 4- 6.8 (10) 47.7 4- 6.2 (11) 25.5 ~ 9.2 (10)

• 3.3 (8) + 3.4 (5) • 2.8 (6) ~= 2.0 (6)

83.7 • 1.9 (12)

78.7 • 1.8 (3)

77.8 -V 4.2 (12)

69.3 4- 1.2 (3)

70.2 4- 3.2 (16)

67.5 -4- 2.5 (4) 59.0 4- 1.7 (9) 52.3 • 1.9 (7) 37.3 4- 2.4 (3) 28.0 4- 2.7 (3) 26.7 4- 4.0 (4) 23.5 • 1.5 (2)

55.9 • 3.6 (13) 41.2 --t-4.2 (12) 13.0 4- 2.7 (13)

17.2 • 1.1 (6) 9.6 :[: 4.2 (5) 10.3 -k 3.0 (3)

a The number in parentheses indicates the number of data points from which the mean and standard error of the mean were computed. The test solution contained 70 mM K +, 2 mM ATP 2-, 15 mM CP 2-, 15 units/cc C2K, 1 • 10-~ M H+; varying amounts of imidazole chloride to keep the ionic strength constant at 0.15 M, and C1- as the major anion. t o i n d e p e n d e n t sites. T h e s i m p l e s t e q u a t i o n d e s c r i b i n g t h e d a t a is t h e H i l l [9] e q u a t i o n w h i c h c a n be e x p r e s s e d a s : ( l~

~176 ) 100-o/o T -----n log10 [Ca 2§ - - logloQ

w h e r e ~ T is t h e p e r c e n t a g e o f m a x i m u m t e n s i o n , n a n d Q a r e c o n s t a n t s , a n d [Ca ~+] refers t o t h e Ca 2+ c o n c e n t r a t i o n of t h e b a t h i n g s o l u t i o n . B o t h t h e s k e l e t a l a n d c a r d i a c d a t a , d i s p l a y e d as m e a n s o f p e r c e n t a g e s of m a x i m u m t e n s i o n ~= S.E.M. are s h o w n i n T a b l e 1. T h e l e g e n d o f Fig. 1

Tension in Disrupted Cardiac Cells

199

gives the n and Q values of the continuous computed curves as determined from fitting the data on a Hill plot [3]. I f certain assumptions are made about the coupling of molecular mechanisms responsible for tension development, values of n greater t h a n 1 can give information about the existence and degree of eooperativity in interactions of Mg ~+, Ca 2+, and the contractile proteins. A discussion of the implications of the value of n is discussed in more detail in a separate paper [3]. As can be seen in Fig. 1 the data for mammalian cardiac muscle and frog shows the same relationship between Ca ~+ concentration and isometric tension for pMg ~ 4.3 and pMg ~ 3. As the concentration of Mg ~+ increases, the pCa~+-versus-tension curve shifts in the direction of higher concentrations of Ca ~+ and its slope declines (i. e., there is less change in tension for a given change in concentration of Ca~+). Maximum tension was not found to be significantly affected b y Mg ~+ concentration.

Discussion Similarity in Sensitivity to Ca 2+ O/ Cardiac and Skeletal Muscle. One striking conclusion t h a t can be drawn from these data is t h a t the relationship between Ca 2+ and Mg 2+ concentrations and percentage of m a x i m u m tension under the specified ionic conditions is quantitatively the same for both skinned frog skeletal muscle fibers and mechanically disrupted r a t ventricular muscle fibers. This is an important finding because it indicates similarity in the molecular mechanisms responsible for the Ca2+-activation in the two types of muscles. Although actin and tropomyosin have not been shown to be chemically very different between types of striated muscles and species [10], Ebashi, et al. [5] observed in skeletal muscle troponin an affinity for Ca 2+ three times greater t h a n t h a t in cardiac muscle troponin. From our data we were not able to determine a difference in Ca2+-aetivation of the muscle fibers.

EHect of Mg 9+. The other conclusion t h a t can be drawn from these data is t h a t Mg ~+ has a striking effec~ on the Ca2+-activation of m a m m a Nan cardiac muscle. As shown in Fig. t, the effect of concentration of Mg 2+ on Ca~+-activated tension in cardiac muscle is essentially the same as in skeletal muscle. The shifting and change in shape of the pCa-tension curve as a function of concentration of Mg 2+ might be due to an effect on the affinity and/or degree of interaction of Mg2+-binding sites. Unfortunately, there have not been a n y studies characterizing the effects of Mg ~+ as separate from MgATP 2- and A T P a- on Ca2+-activated actomyosin ATPase activity and on Ca~+-binding to the contractile proteins; thus it is impossible to relate the effects of Mg 2+ on Ca~+-activat-

200

W. G. Kerricl~ and S. Donaldson

ed tension t o a n y specific protein. However, Mg ~+ has been shown to bind to actin in the mM range [15] a n d to change the eleetrophoretic mobility of t r o p o n i n in the m M range [1]. Ca ~+ induces conformational changes in the calcium-binding c o m p o n e n t of troponin, a n d these conformational changes can be affected b y Mg 2+ in the mlVI range [11]. Thus it is possible t h a t Mg ~+ m a y act on proteins of the thin filament which are chemically similar in the two types o f muscle. The possibility t h a t Mg ~+ affects chemically different molecules, such as myosin light chains f r o m the two muscle types, in exactly the same m a n n e r seems unlikely. The exact n a t u r e of the interaction of Ca ~+ a n d l~g ~+ a n d the role o f Mg 2+ in tension generation of cardiac muscle requires further study. I t is striking, however, t h a t the Ca ~+ activation of tension generation is so similar a n d similarly affected b y Mg 2+ in amphibian skeletal a n d m a m m a l i a n cardiac muscle. This result was u n e x p e c t e d a n d raises questions as to the functional significance of differences in contractile proteins in the two t y p e s of muscle. This project was supported by U.S. Public Health Service Grants HL-13517, ttL-i7373, AM-17081, 1~S-08384, ~vU-00369, NU-5004, and FR-00374 from the National Institutes of Health and by a grant from the Washington State Heart Association.

References 1. Chowrashi, P., Kaldor, G. : Studies on the electrophoretic mobility of troponin in the presence of Ca2+, Mg2+ and (MgATP2-). Proc. Soc. exp. Biol. (N.Y.) 138, 969--972 (1970) 2. Conway, E. J. : Nature and significance of concentration relations of potassium and sodium ions in skeletal muscle. Physiol. Rev. 87, 84--131 (1957) 3. Donaldson, S. K. B., Kerrick, W. G. L.: Characterization of the effects of Mg2+ on Ca~+ and Sr2+-activated tension generation of skinned skeletal muscle fibers. J. gen. Physiol. (in press, 1975) 4. Ebashi, S., Endo, M.: Calcium ion and muscle contraction. Progr. Biophys. molec. Biol. 18, 123--183 (1968) 5. Ebashi, S., Endo, iV[., 0htsuki, I. : Control of muscle contraction. Quart. Rev. Biophys. 2, 35--384 (1969) 6. Ebashi, S., Kodama, A., Ebashi, F. : Troponin I. Preparation and physiological function. J. Biochem. (Tokyo) 64, 465--477 (1968) 7. Fabiato, A., Fabiato, F.: Excitation-contraction coupling of isolated cardiac fibers with disrupted or closed sarcolemmas: calcium dependent-cyclic and tonic contractions. Circular. Res. 81, 293--307 (1972) 8. Hellam, D. C., Podolsky, R. J.: l%rce measurements in skinned muscle fibres. J. Physiol. (Lond.) 200, 807--819 (1969) 9. Hill, A. V.: The combinations of hemoglobin with oxygen and with carbon monoxide. Biochem. J. 7, 471--480 (1913) 10. Katz, A. M. : Contractile proteins of the heart. Physiol. Rev. 50, 63--158 (1970) 11. Kawasaki, Y., van Eerd, J.: The effect of t~Ig~+ on the conformation of the Ca++-binding component of troponin. Biochem. biophys. Res. Commun. 49, 898--905 (1972)

Tension in Disrupted Cardiac Cells

201

12. Kerrick, W. G. L., Best, P. M. : Calcium ion release in mechanically disrupted heart cells. Science 188, 435--437 (1974) 13. Kerriek, W. G. L., Donaldson, S. K. B.: The effects of Mg2+ on submaximum Ca2+-activated tension in skinned fibers of frog skeletal muscle. Biochim. biophys. Acta (Amst.) 275, 117--122 (1972) 14. Kerrick, W. G. L., Krasner, B.: A method for mechanically skinning muscle fiber of mammals. J. app. Physiol. (in press) 1975 15. Martonosi, A., Molino, C. M., Gergety, ft.: The binding of divalent cations to aotin. J. biol. Chem. 289, 1057 (1964) 16. Natori, R. : The proper~y and contraction process of isolated myofibrfls. Jikei reed. J. 1, 119--126 (1954) i7. Podolsky, R. J. : Membrane systems in muscle cells In: Aspects of cell motility. Symposia of the Society for Experimental Biology, No. XXII. New York, New York: Academic Press 1968 18. Polimeni, P. I.: Ionic distribution in rat ventricle. Physiologist 162, 424 (1973) (abstract) 19. Polimeni, P., Page, :E. : Magnesium in heart muscle. Circular. Res. 88, 367--374 (1973) 20. Weber, A., ~Iurray, J. M. : Molecular control mechanism in muscle contraction. Physiol. Rev. 582, 612--673 (1973) W. G. L. Kerrick Department of Physiology and Biophysics, University of Washington School of Medicine Seattle, Washington 98195, U.S.A.