Characterization of Cardiac Microsomal Sarcoplasmic Reticulum Prepared from Control and Diabetic Rats
GARY D. LOPASCHUK, SIDNEY KATZ, AND JOHN H. MCNEILL
A method for the preparation of cardiac sarcoplasmic reticulum (SR) from rat heart is described. SR isolated from control and diabetic rats was characterized to determine if differences in calcium transport activity could originate as an artifact of preparation. Electron micrographs of cardiac SR preparations isolated from control and diabetic rats were indistinguishable. The yeild of SR and the mitochondrial membrane contamination were similar in both preparations. These results suggest
that depression
in diabetic
rat cardiac
SR microsomal
due to nonspecific alterations in the membrane preparations Key Words:
Diabetes;
Cardiac sarcoplasmic reticulum;
function
is not
used.
Streptozotocin
INTRODUCTION In recent years, a number changes
in heart function
tion (Froelich et al., 1983; were
of studies have attempted to alterations
in cardiac
et al., 1978; Alto and Dhalla, Ganguly
isolated
from
was measured.
et al., 1983). cardiac
Cardiac
1981; Penpargkul
In the majority
ventricles
SR isolated
to causally relate pathological
sarcoplasmic
reticulum
of these studies,
and their ability to actively from diabetic
(SRI func-
et al., 1981; Lopaschuk SR microsomes
sequester
rats has been shown
calcium
by a number
of investigators
to have a depressed rate of calcium uptake activity (Penpargkul et to controls. This al., 1981; Lopaschuk et al., 1983; Ganguly et al., 1983) compared defect has been implicated as a possible mechanism by which overall cardiac function is depressed
in chronically
diabetic
rats.
Recently, there has been concern raised about the comparison of calcium uptake activity in cardiac SR microsomes of control and diabetic rats; it has been suggested that nonspecific
artifactual
assay procedures. isolate rat cardiac isolation
of SR from
or the degree
differences
The concerns SR have been
the hearts of other
of mitochondrial
may also lead to error
may be introduced
during
the isolation
and
have arisen, in part, because methods used to adapted from methods previously used for the species.
and SL contamination
in interpretation
Differences between
of experimental
in vesiculation
of SR,
the two preparations,
results.
We therefore
have
From the Division of Pharmacology and Toxicology, Faculty of Pharmaceutical Sciences, University of British Columbia, Vancouver, B.C., Canada. Address reprint requests to: Dr. J.H. McNeill, Division of Pharmacology and Toxicology, Faculty of Pharmaceutical Sciences, University of British Columbia, Vancouver, B.C., Canada, V6T lW5. Received and accepted April, 1983.
199 Journal of Pharmacological
Methods
0 1983 Elsevier Science Publishing
10, 199-206 (1983) Co., Inc., 52 Vanderbilt
Avenue, New York, NY 10017
200
C. D. Lopaschuk et al.
undertaken a study to characterize cardiac SR prepared from control and diabetic rats to determine the validity of using this experimental protocol. METHODS Female Wistar rats (Charles River Canada, Montreal, Canada) were used. Diabetes was induced by a single i.v. injection of streptozotocin (50 mg/kg) dissolved in citrate buffer (pH 4.0). Control rats were injected with citrate buffer alone. Food and water were provided ad libitum throughout the study period (42 days). At the end of the study period, the animals were stunned by a blow to the head and the hearts were removed. Blood was collected in heparinized tubes and centrifuged at 3000 times gravity for 5 min. Serum was assayed for insulin by a radioimmunoassay method (Becton-Dickinson Insulin Radioimmunoassay Kit (1251)), and for glucose utilizing the glucose oxidase method (Sigma Glucose Kit). Cardiac
Sarcoplasmic
Reticulum
Ventricles were excised from hearts, weighed, and placed in ice-cold Tris maleate buffer, pH 6.8. All procedures were carried out at 4°C. Cardiac microsomes enriched in SR were then prepared by a modification of the method of Sumida et al. (1978). Ventricles were homogenized in 15 ml of 10 mM Tris maleate, pH 6.8, with a Teflon pestle for 15 set at 1500 r.p.m.. The homogenate was then centrifuged at 4000 times g for 10 min, and the supernatant passed through cheesecloth. This supernatant was then centrifuged at 40,000 times g for 80 min. The resulting pellet was then resuspended in IO mM Tris maleate, pH 6.8, containing 0.6M KCI and centrifuged at 40,000 times g for 80 min. The final pellet was suspended in 10 mM Tris maleate containing 40% sucrose, quick-frozen in 2-methylbutane on dry ice, and stored at -70°C until use. ATP-dependent calcium uptake was measured by the method of Tada et al. (1974), with a few modifications. Oxalate-facilitated calcium uptake was determined in an incubation medium containing 20-50 kg of microsomal sarcoplasmic reticulum protein, 40 mM histidine hydrochloride, pH 6.8,5 mM MgC12 110 mM KCI, 5 mM Tris-ATP, 2.5 mM Tris-oxalate, and CaC12 containing 45CaCI,. The desired free calcium concentration was maintained by the addition of ethylene glycol bis(B-aminoethyl ether)-N,N’-tetraacetate (EGTA) and the free calcium concentrations present were determined by the equations of Katz et al. (1970). Samples were preincubated for 7 min at 3O”C, and the reaction was started by the addition of 45CaC12. After 5 min, the reaction was terminated by filtering an aliquot of the reaction mixture through a Millipore filter (HA 45, Millipore). The filter was then washed twice with 10 ml of 40 mM Tris-Cl, pH 7.2, dried, and counted for radioactivity in Aquasol (New England Nuclear) by using standard liquid scintillation counting techniques. (Ca2+ -Mg2+)ATPase activity was measured spectrophotometrically utilizing a coupled enzyme assay (Watterson et al. (1976). The incubation medium was similar to that used in the Ca2+ transport studies except that 0.22 mg phosphoenolpyruvate, 0.28 mg NADH, 7 units lactate dehydrogenase, and 42 units pyruvate kinase were present instead of Tris-oxalate. The incubation medium (final volume 1 ml) was equilibrated at 30°C in a Beckman’-24 spectrophotometer. Sar-
Rat Cardiac Sarcoplasmic Reticulum coplasmic reticulum (2-10 kg) was added to the cuvette and preincubated for 5 min. Following the Ca2+ addition, the decrease in absorbance at 340 nm was monitored. Cytochrome c oxidase was assayed spectrophotometrically by the method of Wharton and Tzagaloff (1967). The decrease in absorbance at 500 nm was used as an index of the rate of oxidation of ferrocytochrome C. To each of two cuvettes was added 100 I_LI 100 mM potassium phosphate buffer, pH 7.0, 70 )*I 1% ferrocytochrome c, and 0.83 ml H20. Ferrocytochrome c had previously been reduced with ascorbate and dialyzed extensively. To the blank cuvette was added 10 ~1 0.1 M potassium ferrocyanate to oxidize the ferrocytochrome present. Following equilibration at 37”C, the reaction was initiated by adding 10 ~1 of cardiac SR (approximately 5 pg protein) to the reaction cuvette. The decrease in absorbancy was measured continuously at 550 nm. For the electron microscopic studies, a 0.5 ml suspension of control and diabetic cardiac SR containing approximately 100 kg of protein was centrifuged at 40,000 times g for 40 min. The resultant pellet was fixed in 5 ml 2% glutaraldehyde, 0.06 M cacodylate buffer, pH 7.4. The SR pellet was then stored in 7% sucrose solution at 4°C until further processing. The fixed tissue was processed using standard electron microscopic procedures. Trichloracetic acid precipitable SR protein was assayed by the standard Lowry (1951) protein assay. RESULTS The Chemically-induced
Diabetic Rat Model
During the first three days following injection, approximately 10% of the streptozotocin-injected rats died, probably as a result of hypoglycemic coma due to massive insulin release. The condition of the rats stabilized after three days. Animals which became diabetic (90% of the remaining streptozotocin-injected rats) had an elevated urine glucose (>2%) throughout the study period. As reported previously (Lopaschuk et al. 1983), serum insulin levels, measured at the time of sacrifice, were significantly lower in the diabetic rats. Serum glucose levels were markedly increased in diabetic rats, and weight gain was significantly lower in the diabetic rats as compared to controls. During the study period, the streptozotocin-injected rats displayed a number of symptoms associated with diabetes including polyphagia, polydypsia, and polyuria. Characterization Rats
of Cardiac Sarcoplasmic Reticulum
from Control and Diabetic
The yield of SR obtained from control and diabetic rats is indicated in Table 1. Although the ventricle weights were lower in the diabetic rats, the yield of SR per g of wet ventricle was similar. SR microsomes isolated from control and diabetic rat ventricles were indistinguishable as determined by electromicroscopy, as shown in Figures 1 and 2. Both samples contained an equal number of heterogenously sized vesicles. In these preparations, at 2 ~J,Mfree Ca*+, the rate of calcium transport
201
202
G. D. Lopaschuk et al. TABLE 1 Yield of Cardiac Sarcoplasmic Reticulum from Control and Diabetic Rats YIELD
OF
SARCOPLASMIC RETICULUM CONDITION
Control (n = 9) Streptozotocin-treated
SARC~P~~SMIC WET
1.05 t 0.32a 0.89 5 0.15
RETICULUM/G
VENTRICLE
TISSUE
G)
(MC)
(MC)
@I = 7)
WEIGHT
1.09
2
0.06
0.91 -c 0.07
0.96
0.97
a Results are expressed as the mean 2 S.E.
FIGURE 1. Electron micrograph of cardiac sarcoplasmic reticulum obtained from t:0fl ttrol rats. Sarcoplasmic reticulum microsomes were prepared as described in Methods. Tisisue preparations are shown magnified 19,000 times.
Rat Cardiac Sarcoplasmic Reticulum
FIGURE 2. Electron micrograph of cardiac sarcoplasmic reticulum obtained from six-week diabetic rats. Diabetes was induced by a single i.v. injection of 50 mg/kg streptozotocin. Sarcoplasmic reticulum microsomes were prepared as described in Methods. Tissue preparations are shown magnified 19,000 times.
in control
and diabetic
rat SR was 31.4
nmol/mg/min, respectively. Various incubation conditions
were
? 3.7 (S.D.)
used when
nmollmgimin,
measuring
calcium
and 20.9 uptake
2 2.4 activity
in cardiac SR. The length of the preincubation time was found to not affect the rate of calcium uptake. Similarly, it was found that, up to 20 min, the length of the incubation time did not affect the rate of calcium uptake; between 2 and 20 min, calcium uptake was linear in both control and diabetic rats. In these experiments the amount of SR protein used (36 pg/O.5 ml incubation volume) was kept constant throughout the study. Using previously determined ATP hydrolysis values (Lopaschuk et al., 19831, we found that in no incubation sample was more than 7% of the ATP utilized, and end product inhibition was therefore of no consequence.
203
204
G. D. Lopaschuk et al. TABLE 2 Marker Enzyme Assays on Cardiac Sarcoplasmic Reticulum from Control and 42-day Diabetic Rats ASSAP
CONTROL
Cytochrome c oxidase activityb Azide-sensitive Ca’+-transport activity Azide-sensitive (Ca”-Mg’+)-ATPase activity Ouabain-sensitive (Ca’+-Mg’+)-ATPase activity
53.0 + 5.9 N.D. N.D. N.D.
DIABETIC
49.4 t
3.7
N.D.
N.D. N.D.
Diabetes was induced by a single i.v. injection of 50 mg/kg streptozotocin. N.D. = not detectable. a Cytochrome c oxidase activity is expressed as the nanomole of cytochrome c oxidized per mg SR per min. Ca2+ -transport activity is expressed as the nanomole of Ca’+ transported per mg SR per min. ATPase activity is expressed as the nanomole of ATP hydrolyzed per mg per min. b Result is the mean ? S.E. of five control and five diabetic SR preparations.
To further characterize the cardiac SR preparations, various marker enzyme activities were determined (Table 2). The degree of mitochondrial membrane contamination, as indicated by cytochrome c oxidase, was the same in both control and diabetic rat cardiac SR. This mitochondrial contamination did not contribute to SR calcium accumulation, as determined by the absence of any azide-sensitive calcium transport activity. Also, mitochondrial and sarcolemmal contamination did not appear to contribute to ATP hydrolysis measurements, as determined by the absence of azide-sensitive (Ca’+-Mg2’)-ATPase activity.
DISCUSSION
Before inferences can be drawn concerning the effects of diabetes on cardiac SR function it is necessary to confirm that 1) the diabetes induced chemically in our laboratory is an appropriate model for the study of diabetes, and that 2) cardiac SR preparations derived from control and diabetic animals do not differ in certain critical parameters. The validity of using the chemically-induced diabetic model has been previously documented (Hoftiezer and Carpenter, 1973; Junod et al. 1969) and will therefore not be discussed here. Our interest in this study was to determine similarities and differences in cardiac SR preparations obtained from control and diabetic rats. The methodology used to prepare microsomal cardiac SR has been extensively employed by many laboratories, including our own. The microsomal preparation has also been thoroughly characterized by other investigators (for a review see Jones and Besch, 1979). It is necessary, however, to identify any qualitative differences in SR preparations obtained from control and diabetic rats. Specifically, we were interested in determining if the degree of mitochondrial membrane contamination was altered in the SR prepared from diabetic rats. Also investigated were possible ultrastructural differences, namely the degree and size of microsomal SR vesicles formed.
Rat Cardiac Sarcoplasmic Reticulum
Mitochondrial contamination of our SR preparations was determined by measuring the marker enzyme cytochrome c oxidase. As shown in Table 2, the degree of mitochondrial contamination, within the limits normally found in cardiac SR prepared by this procedure (Jones and Besch, 1979) is not altered in cardiac SR obtained from diabetic rats. In support of our data, a recent report by Ganguly et al. (1983) has also determined that the degree of mitochondrial contamination is similar in SR derived from either control or diabetic rat hearts. The mitochondrial membrane contamination in our SR preparations does not appear to contribute to microsomal calcium uptake. An apparent depression in cardiac SR calcium uptake may simply reflect a smaller number of intact vesicles, compared with unsealed membranes. Similarly, differences in vesicle size may alter the apparent ability of the SR to transport calcium. As shown in Figures 1 and 2, however, the degree of vesiculation was similar in both control and diabetic SR preparations. These date are supported by an earlier finding which shows that any change in the ability of the SR to accumulate calcium was accompanied by changes in calcium-sensitive ATPase activity (Lopaschuk et al. 1983). If an apparent depression in SR calcium uptake was due to a decrease in the ratio of sealed to unsealed vesicles, or of inside-out to rightside-out vesicles, then calcium ATPase activity would not be concomittantly depressed. Another parameter, used to determine if the isolation procedure altered the characteristics of the SR derived from diabetic rat hearts, was the measurement of the yield of SR. Since diabetic rat hearts were smaller, the yield of SR per ventricle was lower in diabetic rats compared to control rats. However, the yield of SR per g weight of ventricle was found to be the same in diabetic and control rats (Table 1). From this study it can be concluded that the method described is a suitable one for measuring calcium uptake in SR prepared from rat heart. Furthermore, the depression in the ability of diabetic rat cardia SR to transport calcium is not an artifact of preparation. The use of this SR preparation may therefore be helpful in uncovering possible mechanisms involved in diabetic rat cardiac dysfunction. This work was supported by grants from MRC(C), the Canadian Heart Foundation, Columbia Branch of the Canadian Diabetes Association.
and the British
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