Estimating the Functional Capabilities of Sarcoplasmic Reticulum in Cardiac Muscle

Transcription

1 Estimating the Functional Capabilities of Sarcoplasmic Reticulum in Cardiac Muscle CALCIUM BINDING By R. John Solaro and F. Norman Briggs ABSTRACT A method was developed to estimate the amount of calcium which can be bound to the sarcoplasmic reticulum of the dog heart. Incubation conditions that permitted calcium oxalate uptake (steady-state filling or uptake rate) to be used as a marker for sarcoplasmic reticulum vesicles in homogenates and microsomal fractions were developed. By dividing the values for peak steady-state filling of a cardiac homogenate by those for peak steady-state filling of a sarcotubular-enriched fraction, we obtained a sarcoplasmic reticulum-homogenate ratio with units of mg sarcoplasmic reticulum X P/g wet heart, where P is the unknown fractional purity of the microsomal fraction. The mean value for the sarcoplasmic reticulum-homogenate ratio obtained from the steady-state filling studies was 6.9 mg sarcoplasmic reticulum X P/g wet heart. Similar values, 6.5 and 7.1 mg sarcoplasmic reticulum x P/g wet heart, were obtained when the rate of calcium oxalate uptake was used as the functional parameter for calculation of the sarcoplasmic reticulum-homogenate ratio. Evidence that sarcoplasmic reticulum vesicles in the homogenate are functionally the same as those in the isolated fraction was obtained. Calcium binding by the sarcotubule fraction was measured by either a spectrophotometric (murexide) or a Millipore filtration technique. Multiplication of the sarcoplasmic reticulum homogenate ratio by the amounts of calcium bound by sarcoplasmic reticulum vesicles (nmoles calcium/[mg sarcoplasmic reticulum x P]) provided an estimate of the ability of the cardiac sarcoplasmic reticulum to bind calcium. Application of this method indicated that the sarcoplasmic reticulum could bind nmoles calcium/g wet heart at 10~ 5 Mfree calcium and 170 nmoles calcium/g wet heart at lo^mfree calcium. KEY WORDS heart calcium uptake rate excitation-contraction coupling dog homogenate of heart oxalate Estimating the amount of sarcoplasmic reticulum in cardiac muscle is difficult because of the lack of a specific marker and because a procedure for the quantitative isolation of a pure fraction has not been developed. Estimates made from electron micrographs (1, 2) provide information only on the volume of muscle occupied by the sarcoplasmic reticulum. Before functional information can be derived from such data, it is necessary to know the mass that such volumes represent and the specific activity of the function under consideration, e.g., the rate of calcium uptake per mass of sarcoplasmic reticulum. From the Department of Physiology, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia 23298, and the Department of Physiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania This investigation was supported by U. S. Public Health Service Grant HL06782 from the National Heart and Lung Institute and grants from the Richmond Area and the Virginia State Heart Associations. Received November 29, Accepted for publication January 16, Cmuktion Research. VoL XXXIV. April 1974 This paper presents a method for estimating the capability of the sarcoplasmic reticulum within a muscle to accumulate calcium. This method can also be used to estimate the rate at which the sarcoplasmic reticulum in muscle can accumulate calcium. In the past, estimates of these capabilities have involved uncertainties about the amount of sarcoplasmic reticulum in muscle and about the calcium uptake rates and binding characteristics of isolated sarcotubule fractions of uncertain purity (3-5). The value of such estimates is compromised by these uncertainties. The capability of the sarcoplasmic reticulum in muscle to accumulate calcium can be estimated without isolation of pure fractions and without knowing the amount of sarcoplasmic reticulum in muscle. The amount of calcium that muscle sarcoplasmic reticulum can bind is determined by the amount of calcium that can bind to an isolated, but not necessarily pure, sarcoplasmic reticulum fraction and the amount of that fraction in the muscle. Under these conditions binding to the sarcoplasmic reticulum fraction is expressed as nmoles/(mg sar- 531

2 532 SOLARO, BRIGGS coplasmic reticulum X P), where P represents the unknown purity of the sarcoplasmic reticulum. The amount of the sarcoplasmic reticulum X P fraction in muscle can be estimated if a sarcoplasmic reticulum characteristic (marker) can be found which can be measured in a homogenate of the muscle. Then the sarcoplasmic reticulum-homogenate ratio can be calculated with the units of mg sarcoplasmic reticulum x P/g muscle. The product of the binding data and the sarcoplasmic reticulum-homogenate ratio, nmoles Ca mg_sr_x_p _ nmoles Ca mg SR X P g muscle g muscle (1) gives the amount of calcium bound by the sarcoplasmic reticulum in muscle. Application of this method requires a method for measuring the sarcoplasmic reticulum-homogenate ratio. Under specific conditions calcium oxalate uptake rates or steady-state uptake levels can be used to determine this ratio. Methods PREPARATION OF CARDIAC SUBCELLULAR FRACTIONS Homogenates and fractions enriched in sarcotubule material were prepared from dog heart muscle as previously described (6). Approximately 50 g of canine cardiac left ventricle was homogenized for 40 seconds in four volumes of 0.3M sucrose and lomm imidazole at ph 7.0 (extraction solution) with a Sorvall Omnimixer set at high speed. A portion of this homogenate was saved, and the balance was centrifuged at 17,000 g for 20 minutes. The supernatant fraction was strained and spun at 34,000 g for 20 minutes. The pellet was resuspended in 0.6MKC1 and 10 mm imidazole at ph 7.0 and centrituged at 198,000 g for 20 minutes. The pellet, termed fragmented sarcoplasmic reticulum, was resuspended in the extraction solution to a protein concentration of 4-8 mg/ ml. CALCIUM UPTAKE AND BINDING EXPERIMENTS Calcium uptake rates, steady-state filling (calcium oxalate capacities), and calcium binding were measured using 45 Ca. Reactions were started by adding the fraction under study, homogenate or fragmented sarcoplasmic reticulum, and stopped by Millipore (0.45/A) filtration- Calcium binding to cardiac fragmented sarcoplasmic reticulum preparations was also estimated from the difference spectrums of murexide (4, 7) using a Zeiss dual-beam spectrophotometer. Specific incubation conditions are given in Results. Buffer, MgCl 2, KC1, and protein were present in both 3-ml cuvettes, and the temperature was maintained at 25 C. After adding 10 //.liters of 100 mm adenosine triphosphate (ATP) to one cuvette, 10 /xliters of 66.7 mm CaCl 2 were added to the same cuvette. Sarcoplasmic reticulum-bound calcium was calculated from the difference in optical density at 470 nm produced by such an addition of calcium in the presence and the absence of protein. The reference isosbestic wavelength was 507 nm. ASSAYS Free calcium ion concentration was calculated using an apparent stability constant for the calcium-ethyleneglycol bis(/3-aminoethylether)-n, N'-tetraacetic acid (EGTA) reaction of 1.1 X lo^" 1 at ph 7.0 (8). The effect of calcium binding to ATP on the above calculation was determined by a method patterned after that of Briggs and Fuchs (9). Contaminant calcium present in buffers and fragmented sarcoplasmic reticulum preparations was determined by atomic absorption spectroscopy using perchloric acid digests of protein. Protein concentrations were determined by the method of Lowry et al. (10). SOLUTIONS AND REAGENTS All solutions were prepared with distilled deionized water. ATP was a product of P-L Biochemicals, creatine phosphate was obtained from Sigma, and EGTA and imidazole were products of Eastman Organic Chemicals. All other chemicals were Mallinckrodt analytical grade reagents. Results Determination of the sarcoplasmic reticulum-homogenate ratio requires a marker for the sarcoplasmic reticulum vesicles and the ability to quantify this marker in homogenate and fragmented sarcoplasmic reticulum preparations. We investigated the possibility of using calcium oxalate uptake as a sarcoplasmic reticulum marker. If the magnitude of caicium oxaiate uptake by the sarcoplasmic reticulum can be made large relative to calcium binding to other organelles (myofibrils, sarcolemmal fragments, or mitochondria) in homogenates or fragmented sarcoplasmic reticulum fractions, then the magnitude of accumulation of calcium oxalate can be used as a quantitative marker for sarcoplasmic reticulum vesicles. The initial problem, therefore, was to minimize calcium binding by potential contaminants and maximize calcium binding to the sarcoplasmic reticulum. Figure 1A demonstrates the well-known ability of 10 mm azide to block calcium accumulation by mitochondria (ll) without significantly affecting the ability of the sarcoplasmic reticulum to accumulate calcium. Azide when added to the whole homogenate (Fig. IB) produced a small effect on calcium accumulation. Although oxalate augments the rate and the magnitude of calcium uptake by the sarcoplasmic reticulum (12), we attempted to define rather Circulation Research, VoL XXXIV, April 1974

3 CALCIUM BINDING BY CARDIAC SARCOPLASMIC RETICULUM r 20 IS 10! TIME IN MINUTES TIME IN MINUTES FIGURE 1 Effect ofazide on calcium uptake by cardiac mitochondria, fragmented sarcoplasmic reticulum and homogenates. Calcium uptake by cardiac fragmented sarcoplasmic reticulum (0.15 mg/ml), mitochondria (0.3 mg/ml), and homogenates (0.003 g heart/ml) was determined at 37 C in an incubation solution consisting of 5 mm ATP, 5 mmmgch, loomukcl, 18 mm imidazole at ph 7.0, 10 mm potassium oxalate, 0.2 mm EGTA, 0.18 mm CaCl2. and 0.05 /xc 4S CaCl 2 lml. A: Fragmented sarcoplasmic reticulum alone (open circles) and in the presence of 10 mm sodium azide (solid circles); mitochondria alone (open triangles) and in the presence of 10 mm sodium azide (solid triangles). B: Homogenate alone (open circles), and in the presence of 10 mm sodium azide (solid circles). carefully the concentration of oxalate that would maximize these parameters. Figure 2 shows the effect of oxalate concentration on uptake rate and uptake capacity. In each preparation, regardless of parameter, 10 mm oxalate appeared to be an optimal concentration. Concentrations of oxalate greater than 10 mm produced no enhancement of uptake and increased the liability of spurious calcium oxalate precipitation. We tested for extra vesicular calcium oxalate precipitation at 10 mm oxalate by inhibiting all calcium uptake with 10 mm tetracaine (13) and by prolonged incubation of protein-free incubation mixtures. Neither of these procedures resulted in crystallization of calcium oxalate or loss of 45 Ca by Millipore filtration of the incubation mixture. SARCOPLASMIC RETICULUM-HOMOOENATE RATIO Determination from Rate Measurements. Oxalate-dependent rates of calcium uptake by sarcoplasmic reticulum vesicles in homogenates and fragmented sarcoplasmic reticulum fractions can be used to estimate the sarcoplasmic reticulum-homogenate ratio. This estimate requires that the parameter be measured under identical conditions in the fragmented sarcoplasmic reticulum fraction and the homogenate. The measurements of the rate of calcium uptake by the sarcoplasmic reticulum must be made not only with the same magnesium and ATP concentrations, temperature, Circulation Research. Vol. XXXIV, April 1974 ph, and ionic conditions but also at the same free calcium concentration in the homogenate and the fragmented sarcoplasmic reticulum fraction. To facilitate our adherence to this stipulation, we UJ a, 100 r 80 hi s 40! o «-> 20 CAPACITY LJ < DC -? UJ OXALATE CONCENTRATION (mm) FIGURE 2 Effect of oxalate on calcium uptake by cardiac homogenates. Steady-state calcium uptake (capacity) at the indicated oxalate concentrations was measured at 37 C in an incubation solution consisting of 5 muatp, 6 mmcreatine phosphate, 5 mmmg&2, 100 mmkcl, 18 mm imidazole at ph 7.0, 1.6 mm EGTA, 0.64 mm CaCh (Ca x IO^M), 0.05 ixc ^CaCh /ml, 10 mm sodium azide, and g heart/ml. The incubation conditions for rate experiments were the same as those described in Figure 1 except that oxalate concentrations were varied. o < en ^> O

4 534 SOLARO, BRIGGS measured the rate of calcium uptake by the sarcoplasmic reticulum over a range of ionic calcium concentrations. The rate of calcium accumulation by sarcotubular vesicles in homogenates and fragmented sarcoplasmic reticulum preparations was determined in an incubation solution consisting of 10 mm potassium oxalate, 5 mm MgCl 2, 5 mm ATP, 100 mm KC1, 10 mm azide, 18 mm imidazole at ph 7.0, 0.2 mm EGTA, and 0.05 /AC 45 CaCl 2 /ml. To achieve a range of calcium ion concentrations, three initial concentrations of calcium (0.18 mm, 0.14 mm, and 0.10 mm) were used. Following uptake, serial samples were taken at intervals of 5-10 seconds and filtered through Millipore filters. The initial and the final total calcium concentration was measured over each interval. Since EGTA is not accumulated (14), the ionic calcium concentration in the bath at the beginning and the end of each interval could be calculated. The mean ionic calcium concentration over an uptake interval was used when the uptake rate was plotted against calcium ion concentration in Lineweaver-Burke plots. Figure 3 shows the effect of calcium concentration on the uptake rate of the fragmented sarcoplasmic reticulum fraction and the homogenate. The ratio of homogenate to fragmented sarcoplasmic reticulum calcium uptake rate at each free calcium level shown in Figure 3 provides a measure of the sarcoplasmic reticulum-homogenate ratio. However, the W jm% ratio was used to calculate the sarcoplasmic reticulum-homogenate ratio, since V m, is easily identified on the reciprocal plot. Table 1 summarizes the kinetic parameters, K m and V^,, obtained from such plots. The average K m of the calcium uptake rate for the fragmented sarcoplasmic reticulum preparation (1.9 /XM) was not statistically different from that for the homogenate (1.5 /LIM) (0.1 <_P^0.5). Thus, the dependence of rate of calcium uptake on free calcium is the same ii I/Co* FIGURE 3 TABLE 1 Calcium Uptake Parameters of Cardiac Homogenate and Fragmented Sarcoplasmic Reticulum x P Parameter Homogenate HOMOGENATE 1.5 ((jm -') Reciprocal plot of the relation between calcium ion concentration and the rate of calcium uptake by cardiac fragmented sarcoplasmic reticulum (FSR) and homogenate. Calcium uptake by cardiac fragmented sarcoplasmic reticulum (0.075 mg/ml) and homogenate (0.003 g heart/ml) was measured at 37"C. Incubation conditions and determination of calcium ion concentration are discussed in the text. The data was fitted by a linear regression. for sarcoplasmic reticulum vesicles in the homogenate and in the fragmented sarcoplasmic reticulum fraction. By dividing the average V^, obtained in the homogenate experiments (10.95 fimoles/g min" 1 ) by the average V^, obtained in the fragmented sarcoplasmic reticulum experiments (1.54 ju,moles/mg min" 1 ), the sarcoplasmic reticulum-homogenate ratio is 7.1 mg sarcoplasmic reticulum x P/g wet heart. Since the rate of calcium uptake by sarcoplasmic reticulum vesicles was a function of oxalate concentration (Fig. 2) and calcium concentration, the effect of oxalate concentration on the sarcoplasmic reticulum-homogenate ratio was examined, figure 4 shows results of experiments in which rates of calcium uptake by homogenates and fragmented sarcoplasmic reticulum fractions were measured at a constant (saturating) level of ionic calcium with various concentrations of oxalate. The plots can be made to superimpose, indicating a sarcoplasmic re- Sarcoplasmic reticulum- homogenate ratio Fragmented sarcoplasmic reticulum x P K m (calcium vs. rate) V mai (uptake rate) Oxalate capacity 1.5 ± 0.2 /AM ± 0.59 pimoles/g heart min" ± 0.6 /imoles/g heart ± 0.17 i (mg sarcoplasmic reticulum X P mirt ± 0.1 (mg sarcoplasmic reticulum XP) 7.1 mg sarcoplasmic reticulum X P/g heart 6.9 mg sarcoplasmic reticulum X P/g heart Values are means ± SE. Six dog hearts were studied. Circulation Research, Vol. XXXIV. April 1974

5 i 0UJ CALCIUM BINDING BY CARDIAC SARCOPLASMIC RETICULUM 535 ri 10 i.8 CONCENTRATION OX A LATE (mm) i 4 ^. 3 b 4 0 <3 FIGURE 4 Effect ofoxalate concentration on the rate of calcium uptake by cardiac homogenates and fragmented sarcoplasmic reticulum fractions (t"sr). The rate of calcium uptake, at the oxalate concentrations indicated, by cardiac homogenates (open triangles) (0.003 g heart/ml) and fragmented sarcoplasmic reticulum (solid circles) (0.15 mg/ml) was measured at 37 C in 10 mm sodium azide. Otherwise, incubation conditions were the same as those in Figure 1. Uptake rate was calculated from the amount of calcium accumulated in 0.5 minutes by homogenates and in 0.1 minutes by fragmented sarcoplasmic reticulum. ticulum-homogenate ratio of 6.5 mg sarcoplasmic reticulum X P/g wet heart. Determination from Capacity Measurements. The sarcoplasmic reticulum-homogenate ratios were calculated by measuring steady-state filling (calcium oxalate capacity) of sarcoplasmic o o» to to 40 3, UJ z o ^ HOMOGENATE y*,^ g heart/ml Xs*^"* * f g heart /ml /^ i i i i TIME (MINUTES) reticulum vesicles in homogenates and fragmented sarcoplasmic reticulum fractions. To make this measurement, it was necessary to determine the time required to reach steady state and the effect of vesicular concentration on this time and on filling. Figure 5 shows the time required to reach steady-state filling at two concentrations of homogenate and fragmented sarcoplasmic reticulum fraction. The magnitude offillingand the time required to reach steady state are functions of the concentration of vesicular material (Fig. 5). This point is emphasized by the data shown in Figure 6, in which the calcium oxalate capacity is plotted as a function of fragmented sarcoplasmic reticulum and homogenate concentration. We concluded from these data that filling, except for a limited range, depends on the concentration of the fragmented sarcoplasmic reticulum fraction or the homogenate; therefore, filling per se cannot be used to calculate the sarcoplasmic reticulum-homogenate ratio unless a particular level of steady-state extravesicular calcium is specified. This dependence of the sarcoplasmic reticulum-homogenate ratio on the concentration of vesicular material shows that, as fragmented sarcoplasmic reticulum or homogenate concentration increases, the level of extravesicular calcium does not decrease linearly. If filling of sarcoplasmic reticulum vesicles in the homogenate and in the fragmented sarcoplasmic reticulum fraction equally depends on the level of extravesicular calcium at steady-state, then the ratio of sarcoplasmic reticulum capacity to homogenate capacity at any /) ii a. CALC / A'' ^ x FSR mg/ml i i i i TIME (MINUTES) FIGURE 5 Calcium uptake by cardiac homogenates and fragmented sarcoplasmic reticulum (FSR). Uptake was measured at the concentrations ofhomogenate and fragmented sarcoplasmic reticulum shown under the same incubation conditions described in Figure 2. Circulation Research, Vol. XXXIV. April 1974

6 536 SOLARO, BRIGGS >- 6 < 4 (J 2 ^HOMOGENftTE HOMOGENATE (g heart/ml) FSR (mg/ml) o-o FIGURE < Steady-state calcium uptake (capacity) at various concentrations of cardiac homogenates and fragmented sarcoplasmic reticulum(fsli\). Incubation solutions are given in Figure 2. particular extravesicular free calcium concentration should be constant (Fig. 7). In Figure 7 the extravesicular free calcium concentration is plotted as a function of homogenate and fragmented sarcoplasmic reticulum concentration; the plots are superimposed nearly exactly, indicated a sarcoplasmic reticulum-homogenate ration of 6.7 mg sarcoplasmic reticulum-homogenate ratio of 6.7 mg sarcoplasmic reticulum X P/g wet heart for any level of steady-state free calcium. Table 2 summarizes the results of the three approaches used in this experiment to estimate the < a: _l <? 0 HOMOGENATE(g heart/ml) FSR(mg/ml) o o FIGURE 7 Steady-state free calcium at various concentrations of fragmented sarcoplasmic retictdum and homogenate. The ordinate is the extravesicular free calcium concentration in the incubation solution at steady state. I o o I sarcoplasmic reticulum-homogenate ratio. The ratio is nearly the same regardless of the functional parameter (rate or capacity) used as a marker for the sarcoplasmic reticulum vesicles. The average sarcoplasmic reticulum-homogenate ratio (6.8 mg sarcoplasmic reticulum X P/g wet heart) was then used in conjunction with measurements of calcium binding by fragmented sarcoplasmic reticulum preparations to estimate the ability of cardiac sarcoplasmic reticulum to store calcium. CALCIUM BINDING BY THE CARDIAC SARCOPLASMIC RETICULUM To estimate the amount of calcium which can be bound by cardiac sarcotubules, we determined the amount of calcium which is bound per mg sarcoplasmic reticulum X P (Eq. l). Although calcium binding to sarcotubule fractions from canine heart has been reported, these data are not useful because the purity of the preparations was not identical to that of the preparations we used to measure the sarcoplasmic reticulum-homogenate ratio. The same fragmented sarcoplasmic reticulum preparation must be used for the binding determinations and the ratio determinations so that the values for purity in Eq. 1 will cancel. We measured calcium binding by cardiac fragmented sarcoplasmic reticulum fractions by either the dual-beam spectrophotometric technique with murexide as a calcium indicator (4, 7) or by Millipore filtration (3, 5) (Table 3). Binding measured by the spectrophotometric technique (64 nmoles/[mg fragmented sarcoplasmic reucuiuiu /\ ij; wtts sumcniui ii.gi.ci' i».ii». ^..a>. measured by Millipore filtration (49 nmoles/[mg fragmented sarcoplasmic reticulum X P]). This higher binding was probably due to the higher level of free calcium in the murexide experiments. Other investigators (4, 15, 16) have reported fragmented sarcoplasmic reticulum calcium binding values similar to those presented in Table 3. The fragmented sarcoplasmic reticulum calcium binding in Table 2 is, however, higher than that reported by Pretorius et al. (17) (20 nmoles/mg) and by Katz and Repke (5) (26 nmoles/mg). These discrepancies are probably due to different degrees of purity, since incubation conditions were similar in all of these studies. Since cardiac contractile proteins probably operate at less than maximal activation (18), it was appropriate to measure calcium binding at levels of free calcium below those required to fully activate cardiac myofibrils. Therefore, we measured the relation between sarcoplasmic reticulum calcium Circulation Research, Vol XXXIV, April 1974

7 CALCIUM BINDING BY CARDIAC SARCOPLASMIC RETICULUM 537 Summary of Sarcoplasmic Reticulum-Homogenate Ratios TABLE 2 Method of determination Rate ratio at various calcium ion concentrations Rate ratio at various oxalate concentrations Capacity ratio at various steady-state calcium uptakes MEAN ± SE Sarcoplasmic reticulum-homogenate ratio (mg sarcoplasmic reticulum x p/g wet heart) ± 0.2 binding and free calcium using the Millipore filtration technique (Fig. 8). The insert in Figure 8 shows a reciprocal plot of the binding data which indicates that binding is half-maximal at 0.4 fim free calcium. This K m for calcium binding agrees well with that reported by Repke and Katz (16) and by Harigaya and Schwartz (15). Values for binding at 1.0 /AM and 0.1 fim free calcium obtained from four experiments like that shown in Figure 6 are given in Table 3. The data shown in Table 3 for calcium binding per mg fragmented sarcoplasmic reticulum X P were multiplied by the average sarcoplasmic reticulum-homogenate ratio given in Table 2 (6.8 mg fragmented sarcoplasmic reticulum X P/g wet heart). Thus, at 50 fj.u free calcium, dog cardiac sarcoplasmic reticulum can bind nmoles calcium/g wet heart, but at 1 /im free calcium it binds only half of this amount. Discussion The validity of using 45 Ca uptake as a marker for the sarcoplasmic reticulum requires that no other organelle accumulate calcium in amounts that are significant when compared with that accumulated by vesicles of the sarcoplasmic reticulum. Calcium uptake by mitochondria was eliminated from fragmented sarcoplasmic reticulum and homogenate preparations by adding azide to the incubation medium (Fig. 1). Calcium uptake by the fragmented sarcoplasmic reticulum preparation could Calcium Binding by Canine Cardiac Sarcoplasmic Reticulum Method of measurement Free calcium concentration (MM) TABLE 3 not have been significantly influenced by sarcolemmal contamination. Sulakhe et al. (19) have shown that sarcolemmal vesicles, under conditions similar to those in this study, accumulate only 0.19 pimoles calcium/mg, and Table 1 shows that the fragmented sarcoplasmic reticulum accumulates 5.6 fxmoles/mg. Thus, sarcolemmal protein could only account for 3% of the calcium taken up by the fragmented sarcoplasmic reticulum fraction, assuming that the fragmented sarcoplasmic reticulum is pure sarcolemmal vesicles. Therefore, calcium binding by sarcolemmal fragments in the homogenate is trivial compared with that of the sarcoplasmic reticulum. The ratio of sarcolemmal membrane area to sarcoplasmic reticulum membrane area in heart muscle as measured by quantitative electron microscopy is 1 to 4 (2). Since the ratio of sarcolemmal calcium oxalate uptake to fragmented sarcoplasmic reticulum calcium oxalate uptake is 1 to 20 (18), calcium uptake by sarcolemmal vesicles in homogenate could not account for more than 1% of the total uptake. Finally, sarcolemmal calcium transport is half-maximal at 2 X 10~ 5 M free calcium (18), calcium oxalate transport by sarcoplasmic reticulum vesicles is half-maximal at 1.0 x lo^m (Table 1), and calcium binding by the fragmented sarcoplasmic reticulum preparation is half-maximal at 4 X1O" 7 M free calcium (Fig. 8). Thus, at the free calcium levels used in our measurements (3 x 10" 7 M-l x 10" 5 M), sarcolemmal vesicles should ac- Bound calcium (nmoles/[mg sarcoplasmic reticulum x P]) Bound calcium (nmoles/g wet heart) 0 Murexide technique (N - 6) Millipore filtration (N 4) Millipore filtration (N - 4) Millipore filtration (N - 4) ±3 49 ±6 25 ±2 4± Values for bound calcium are means ± SE. N = number of dog hearts studied. 'Sarcotubular calcium binding per g wet heart was calculated using Eq. 1 and a sarcoplasmic reticulum-homogenate ratio of 6.8 mg fragmented sarcoplasmic reticulum/g (Table 2). Circulation Research, Vol. XXXIV. April 1974

8 538 SOLARO, BRIGGS 008 z 1 ; 0O6 I! 1 ^ 004! ~ 0.02 ^ y1 2 3 l/co"(um-') I0" 8 I0' 7 10" 6 IO" S CALCIUM ION CONCENTRATION FIGURE 8 Relation between calcium ion concentration and calcium bound by cardiac fragmented sarcoplasmic reticulum. Binding was determined after a 1 -minute incubation at 25"C in 100 tnmkcl, 20 mid imidazole at ph 7.0, 3 mm ATP, 3 mtt MgCh, 0.05 \ic 45 CaCh/ml, 50 (M CaCh (added + contaminant), and HUEGTA total. The reciprocal plot was fitted by linear regression. cumulate or bind little, if any, calcium. Quantitatively significant calcium binding to myofibrils in the fragmented sarcoplasmic reticulum preparation is unlikely since precautions were taken to dissolve such contaminants and because the magnitude of such binding (1-2 nmoles/mg) (20) is insignificant compared with the 5.5 /xmoles/mg of 45 Ca taken up by the fragmented sarcoplasmic reticulum fraction (Table 1). (Jalcium binding to the myofibrils in the homogenate is even less significant. Calcium uptake by the homogenate derived from 1 g of cardiac muscle amounted to 38.7 fimoles/g muscle (Table 1). The same 1 g of muscle contains about 50 mg of myofibrils (19, 20). During the uptake experiments these myofibrils bind about 0.05 /xmoles of calcium (20), which is an insignifioant fraction of the calcium taken up by the homogenate. Therefore, calcium uptake from a medium containing oxalate, azide, and EGTA can be used to mark and quantify the amount of sarcotubule material in fragmented sarcoplasmic reticulum fractions and homogenates. Since the calcium uptake characteristics (rate or capacity) of the fragmented sarcoplasmic reticulum fraction is used to evaluate the amount of sarcotubule material in the homogenate, it is important that the isolated sarcotubule material in the fragmented sarcoplasmic reticulum fraction represent the same material in the homogenate. (M) 50 JO This point is particularly important because only about 10% of the sarcotubule material is obtained in the fragmented sarcoplasmic reticulum fraction. This 10% estimate is based on the following observations. The oxalate capacity of isolated fragmented sarcoplasmic reticulum was 5.6 /xmoles/mg fragmented sarcoplasmic reticulum, and the yield of fragmented sarcoplasmic reticulum averaged 0.63 mg/g wet heart. If this yield represents all of the sarcoplasmic reticulum vesicles in the homogenate, then the calcium oxalate capacity of the homogenate should be 3-4 /nmoles/g wet heart. However, Table 1 shows that the homogenate capacity for calcium oxalate accumulation averaged 38.7 /umoles/g wet heart. Thus only 10% of the homogenate capacity for calcium oxalate is recovered in the fragmented sarcoplasmic reticulum fraction. A similar conclusion was reached by Ogawa (21) concerning yields of sarcoplasmic reticulum from skeletal muscle. The fact that approximately 70% of the homogenate capacity for calcium oxalate accumulation appears in the myofibrillar pellet which is discarded after the first centrifugation demonstrates that cardiac sarcoplasmic reticulum is lost during isolation and not merely inactivated. If the sarcoplasmic reticulum vesicles freed by homogenization and sedimented during isolation are identical to the sarcoplasmic reticulum vesicles which remain associated with the myofibrils, mitochondria, and other debris in the homogenate, then the functional activity of the sarcoplasmic rencuium vesieies in Luc ^ y in an identical fashion to that of the fragmented sarcoplasmic reticulum. A strong piece of evidence that favors functional similarity of pelleted and nonpelleted sarcoplasmic reticulum vesicles is the fact that the K m for calcium transport was nearly the same whether it was measured with sarcoplasmic reticulum vesicles in the fragmented sarcoplasmic reticulum preparation or in the homogenate (Table 1). Furthermore, the V^for calcium transport by sarcoplasmic reticulum vesicles measured in the homogenate should be identical to that calculated from fragmented sarcoplasmic reticulum measurements multiplied by the sarcoplasmic reticulum-homogenate ratios (Eq. 1). Using a sarcoplasmic reticulum-homogenate ratio of 6.8 mg fragmented sarcoplasmic reticulum X P/g wet heart and a V^of 1.5 ju,moles/(mg fragmented sarcoplasmic reticulum X P) min~' (Table 2), a maximal uptake rate by the sarcotubules in the heart of 10.2 /imoles calcium/g wet heart min~' is obtained. Circulation Research, Vol. XXXJV. April 1974

9 CALCIUM BINDING BY CARDIAC SARCOPLASMIC RETICULUM 539 This value is very close to the average V max (10.9 /xmoles calcium/g wet heart min~') obtained from the homogenate data plotted by the Lineweaver- Burke method (Table 1). Further evidence that pelleted sarcoplasmic reticulum vesicles retain functional similarity may be inferred from data shown in Figures 4 and 7. Figure 4 shows that, by proper scaling, plots of calcium uptake rate by homogenates and fragmented sarcoplasmic reticulum fractions as functions of oxalate concentration (Fig. 7) can be made to superimpose indicating that calcium uptake by sarcotubule material in the fragmented sarcoplasmic reticulum and the homogenate are similar functions of the concentration of calcium. In skeletal muscle, the sarcoplasmic reticulum has been classically regarded as both the source and the sink for the calcium involved in the activating of myofibrils (22). Since evidence has been presented which indicates that superficially bound calcium might be the source of the calcium which activates cardiac myofibrils (23), the role of cardiac sarcoplasmic reticulum as a source for activating calcium is currently under debate. Thus, a determination of the ability of cardiac sarcoplasmic reticulum to bind and store calcium will help to resolve the role of the sarcoplasmic reticulum in the contraction-relaxation cycle of the heart. Using the sarcoplasmic reticulum-homogenate ratio (Table 2) and estimates of calcium storage capacity of fragmented sarcoplasmic reticulum fractions (Table 3), it is possible to estimate this calcium storage capability with Eq. 1. At free calcium levels (20-50 (JLM) at which myofibrillar activation is maximal (20), cardiac sarcoplasmic reticulum can bind nmoles/g wet heart (Table 3). Estimates of the amounts of calcium bound to cardiac myofibrils under the conditions of the fragmented sarcoplasmic reticulum calcium binding experiments have been made in our laboratory (20) and are similar to the estimates of calcium requirements for maximal cardiac activation calculated by Katz (24), i.e., about nmoles/g wet heart. Therefore, the cardiac sarcoplasmic reticulum can bind enough calcium to maximally activate cardiac myofibrils. At about half-maximal activating levels of free calcium (1 /XM), cardiac sarcoplasmic reticulum can bind 170 nmoles calcium/g wet heart; this amount exceeds that required for halfmaximal activation of cardiac myofibrils (22 nmoles/g wet heart) (20). Therefore, our experiments indicate that cardiac sarcoplasmic reticulum can bind more calcium than that required to acti- Circulation Research. Vol. XXXIV. April 1974 vate the myofibrils. Thus, the sarcoplasmic reticulum has the potential to be the source of activating calcium in the heart. Although cardiac sarcoplasmic reticulum can bind the amounts of calcium involved in electrochemical coupling, the question of whether the sarcoplasmic reticulum can bind these calcium ions rapidly enough to account for the relaxation phase of the heart will require the combination of measurements of rapid kinetics of calcium uptake by the fragmented sarcoplasmic reticulum fraction and the sarcoplasmic reticulum-homogenate ratio. References 1. PEACHEY. L.D.: Sarcoplasmic reticulum and transverse tubules of the frog's sartorius. J Cell Biol 25: , PAGE, E., AND MCCALUSTER, L.P.: Quantitative electron microscopic description of heart muscle cells. Am J Cardiol 31: , PALMER, R.F., AND POSEY, V.A.: Ion effects on calcium accumulation by cardiac sarcoplasmic reticulum. J Cen Physiol 50: , MCCOLLUM, W.B., BESCH, H.R., JR.. ENTMAN, M.L., AND SCHWARTZ, A.: Apparent initial binding rate of calcium by canine cardiac relaxing system. 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