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Depressed cardiac sarcoplasmic reticular function from diabetic rats
Journal
of Molecular
and Cellular
Depressed Somsong
Cardiology
Cardiac Penpargkul,
(1981)
13, 303-309
Sarcoplasmic from Diabetic Frederick and James
Reticular Rats
Fein, Edmund Scheuer*
Function H. Sonnenblick
Division
of Cardiology, Defiartment of Medicine, Montejore Hospital and Medical Center and Albert Einstein College of Medicine the Department of Physiology, Albert Einstein College of Medicine, Bronx, New York (Received 23 June 1980, accepted in revised form 19 November 1980) S. PENPARGKUL, F. FEIN, E. H. SONNENBLICK AND J. SCHEUER. Depressed Cardiac Sarcoplasmic Reticular Function from Diabetic Rats. Journal of Molecular and Cellular Cardiology (1981) 13, 303-309. Previous studies have demonstrated impaired contractile performance and delayed relaxation of hearts of diabetic rats. Male and female rats were made diabetic with intravenous streptozotocin and their hearts were studied 4 to 5 or 9 weeks later. Plasma glucose in female diabetics averaged 453 mg/lOO ml and in male diabetics 615 mg/lOO ml. Calcium uptake by isolated sarcoplasmic reticulum in the absence of oxalate were significantly lower in preparations from hearts of diabetic males and females than from controls. Rats pretreated with J-O-methyl glucose before streptozotocin also had normal calcium binding by SR in the absence of oxalate. Sarcoplasmic reticulum Mg2+ ATPase and Gas+-Mgsf (total) ATPase activities were significantly depressed in preparations from the diabetic animals. The results may partially explain the abnormalities in contraction and relaxation previously observed in hearts of diabetic animals. KEY
WORDS:
Calcium;
Streptozotocin;
ATPase;
Cardiac
relaxation;
Diabetes;
Sarcoplasmic
reticulum.
Introduction Previous investigations in experimental animals and in humans indicate that diabetes is associated with a specific cardiomyopathy [S, 14-161. Studies using papillary muscles [3] and isolated perfused hearts [23] from rats with streptozotocin induced diabetes demonstrate abnormalities in heart muscle function. The most prominent finding is a delay in the rate of relaxation. This defect persists in studies in which adequate oxygenation and energy generation appear to be assured, leading to the hypothesis that abnormalities exist in the energy utilizing mechanisms responsible for contraction and relaxation. Malhotra et al. [IO] examined contractile protein function in preparations of hearts of diabetic rats and found depressed Ca2+-activated myosin and actomyosin ATPase activities. These findings could explain abnormal cardiac contraction but not the slowing of relaxation. Since the calcium control by the sarcoplasmic reticulum plays an important role in modulating cardiac relaxation the following studies were conducted. Materials
and
Methods
Male Wistar rats weighing 180 to 200 g were made diabetic by injecting streptozotocin 50 mg/kg dissolved in 0.05 M citrate, pH 4.5 into the tail vein [23]. Control rats from the same initial group were injected with 0.05 M citrate only. Female * Reprint Bronx, New
requests to J. Scheuer, York 10467, U.S.A.
0022-2828/8
l/030303+
07 $02.00/O
Montefiore
Hospital 0
and Medical 1981 Academic
Center,
111 East 210th
Press Inc.
(London)
Street, Limited
304
S. Penpargkul
et al.
Wistars were injected with streptozotocin 60 mg/kg at initial weights of 170 to 200 g. All rats were allowed to ingest glucose in water for the next 24 h and were then fed normal Purina Rat Chow until they were killed. At weekly intervals, blood was drawn from the tail vein of control and diabetic animals for glucose determinations. Body weights were also recorded at weekly intervals to determine the growth rate. Details of blood analyses conducted on this model of diabetes have been outlined previously [IO, 131. Two groups (five to six rats each) of female rats were treated with 1.65 mmol of 3-O methyl glucose (Sigma) immediately before streptozotocin injection. This intervention prevents the diabetogenic effect of streptozotocin [J?, 51. Animals treated with 3-O methyl glucose were killed and studied at the same time as the diabetic female groups. Sarcoplasmic reticulum (SR) were isolated from pools of four to six ventricles as described by Penpargkul [II]. The homogenization was performed in 0.3 M sucrose and 10 mM imidazole (pH 7.4) using a Tekmar homogenizer. The final pellet was suspended in 50 mM KC1 and 10 mM imidazole, pH 6.8 (2 to 3 mg SR/ml). Calcium uptake was studied in the absence and presence of oxalate by the Mcllipore filtration technique. The reaction medium contained 120 mM KCl, 10 FM imidazole (pH 6.8), 5 mM MgCI,, 5 mM Tris-ATP, 7 mM phosphoenolpyruvate, 0.1 mg/ml pyruvate kinase, 5 mM sodium azide, 0.1 mM EGTA, 0.1 mM CaCl, and 0.15 mg SR protein/ml [12]. The study was performed at 25°C. The reaction was started by addition-of the?& For the assay of sarcoplasmic reticulum Mg a+-ATPase and Ca2+-Mg2+-ATPase activities, the reaction medium was similar to that used in calcium uptake. The total volume was 1 to 2 ml containing 0.05 mg SR protein/ml. For the Mg2+-ATPase determination the CaCI, was omitted from the reaction medium. The reaction was started by adding SR or ATP. At 1, 2, 4 and 10 min after starting the reaction, 0.2 to 0.5 ml was removed from the reaction tube and was added to 0.15 ml of 6% HCIO, to terminate the reaction. After centrifugation at 3000 g for 10 min at 2 to 4”C, an aliquot was taken for phosphate determination. Free calcium in the reaction medium was calculated by two different methods, as described by Katz et al. [7], and by Solar0 and Shiner [18] to be 5.4 pmol and 2.94 pm01 respectively. The cytochrome oxidase was determined by the method of Wharton and Tzagoloff [19]. Glucose was determined using an enzymatic method [I]. Inorganic phosphate was analyzed by the method of Fiske and SubbaRow [4]. The SR protein was measured by the method of Lowry et al. [9]. Analysis of sarcoplasmic reticulum preparations from control and diabetic animals were always performed simultaneously as paired samples. Statistical differences between mean values were evaluated by using analysis of variance for paired data [2].
Results
Streptozotocin induced diabetes causes plasma or serum glucose to increase rapidly within 24 h and to remain elevated until the time when rats were killed [IO, 121. Table 1 shows that in the current experiments plasma glucose was markedly elevated at the time of rats being killed in anesthetized animals. Table 1 also demonstrates that body weights, and heart weights were significantly less at the time of killing in
Sarcoplasmic
Reticulum
305
in Diabetes
diabetic animals than in controls, and in the present series of experiments, heart weight/body weight ratios were higher in the diabetic animals. Heart weight was depressed 30% in diabetic males and only 13 o/0 in diabetic females.
TABLE
1. Plasma
glucose,
heart
Body
weight
weight
and body Heart
(9)
Females
(4 to 5 weeks) control (9) diabetic 3-OMGt(l1)
Males
(9 weeks) control diabetic
weight
weight cd
rats
in diabetic
Plasma glucose (mg/lOO ml)
HW/BW b-+&d
(9)
230 f 186 f 238 f
4 7* 12
0.606 0.527 0.630
0.006 0.015* 0.012
2.63 2.84 2.65
f * f
0.003 0.006* 0.004
160 f 453 f 229 *
(9) (9)
447 f 263 f
8 14*
0.975 f 0.038 0.681 & 0.027*
2.19 2.60
f
0.005
170 615
Results are means i S.E. Numbers in parentheses are the number * P < 0.001 compared with control. t 3-O MG = 3-O methyl glucose treated
f j, f
relationships
of paired
f 0.005*
11 55* 20*
f 16 & 55*
studies.
rats.
Preparations of sarcoplasmic reticulum from hearts of females yielded SR protein of 0.73 & 0.07 mg/g in controls versus 0.83 & 0.08 in diabetics, and in males 0.69 & 0.05 mg/g in controls versus 0.63 f 0.06 in diabetics. The values between controls and diabetics were not significantly different. Cytochrome oxidase activity in preparations of sarcoplasmic reticulum indicated a similar degree of mitochondrial contamination (X = 7.1 f 1.3 min-r/mg control, vs. X = 5.4 f 0.4 diabetic). Table 2 gives the values for SR calcium uptake in hearts from female and male diabetic rats. Values for these functions were significantly lower in preparations of hearts of diabetics than from controls both in the presence and absence of oxalate. Rats treated with 3-O methyl glucose had heart and body weights similar to control rats and plasma glucose levels were elevated, but lower than found in diabetic animals. Calcium uptake by SR appeared to be similar to that found in control preparations when oxalate was absent. Calcium uptake was intermediate between control and diabetic where oxalate was present. Table 3 gives values of Mg2+ and Ca2+-Mg2+ activated ATPase from male and female diabetic rat hearts. There were marked depressions in Mg2+-ATPase of sarcoplasmic reticulum from hearts of male and female diabetic animals. SR isolated from hearts of normal female rats tended to have lower enzymatic activites than SR isolated from hearts of normal male rats. The calcium transport in the presence as well as absence of oxalate by the SR from female rat hearts was also lower than that of male rat hearts. Discussion
The present study shows that there are several abnormalities in isolated sarcoplasmic reticulum from hearts of rats made to be diabetic with streptozotocin. These include depressed calcium uptake and diminished Mg2+ and Ca2+-Mg2+-ATPase activities.
(oxalate
Female control Female diabetic Female 3-O MGT Male control Male diabetic
Casf-uptake (nmol/md
bmol/md Female control Female diabetic Female 3-O MGB Male control Male diabetic
(oxalate
Gas+-binding
Ca2+ uptake
2.
uptake
(8)
(8)
(8) (8) (2)
-
36.8 -& 1.9 27.8 f 1.2*
60.1 40.4 51.5 90.9 50.6
6.8 8.03
reticulum
f 7.8 & 9.8*
f f
hearts.
-
-
30 s
2.6
(7) (7)
present)
sarcoplasmic
4.38
32.4 23.6 41.6
f f
15s
by cardiac
(8) (8) (2)
omitted)
and
Results are mean & S.E. Numbers in parenthesis are the number of paired studies. * P < 0.001; t P < 0.01; $ P < 0.05. 7 3-O methyl glucose. n = two preparations of 5 to 6 pooled
TABLE
diabetic
3.5 5.93 3.9 2.5t
* f f
118 & 18 73 & 14s 66 166 f 13 lll-& 13*
44.1 67.5 64.6 49.4
f
1 min
59.4
from
animals
f f
* f
307 5 235 -j= 253 489 -& 346 &
79.8 56.5 98.5 97.4 65.3
4 min
37 39t
34 508
6.3 4.6t
6.9 5.9
649 521 620 940 714
184.0 109.0
129.2 77.5 136.7 18 11t
10 12t
f f
88 553
f 64 & 108
f f
f f
10 min
905 768 824 1142 925
z
-
f f
f f
15 min
134 889
67 148
Sarcoplasmic
TABLE
3. Sarcoplasmic
reticulum
Reticulum ATPase
activities
from
MgZ+-ATPase bWmd Females
1 min
control
diabetic 10 min control diabetic Males
1 min 10 min
control diabetic control diabetic
(5)
(5) (5) (5) (6) (6) (6) (6)
Results are mean + S.E. Numbers in parentheses are the number * = P < 0.001. t = P < 0.01.
3.50 2.18 22.8
f f
0.30 0.30t
i 14.6 f
1.0 1.61
4.38 f 2.50 f 28.9 f 15.8 f
0.30 0.34* 1.9 2.0*
of paired
307
in Diabetes hearts
of diabetic
rats
Ca2+ Mg2+-ATPase (wolbd 3.50 f 2.32 f 24.4 f 16.0 f 4.46 2.79 29.90 17.00
f f f f
0.30 0.237 1.60 1.6Ot 0.31 0.33* 2.2 2.0*
studies.
Studies of sarcoplasmic reticulum from cardiac muscle are always plagued by the possibility that unrecognized impurities of the preparations may result in artifactual conclusions. An inert contaminant that is contained in the preparations of diabetic hearts but not of the control hearts could explain depressions in enzymatic and calcium uptake characteristics of the SR from diabetic hearts. Such a contaminant would be expected to result in uniform differences for various measurements. In the present experiments the degree of depression of activities observed differed depending upon the measurement. For instance SR from male diabetic hearts showed 25,33 and 45% depressions in Gas+ uptake without oxalate, Ca2+ uptake with oxalate, and Mg2+-ATPase activity at 1 min respectively, suggesting that contamination with inert material was not responsible for these findings. The most likely active contaminant would be mitochondrial fragments. Measurements of cytochrome oxidase activity were made and no difference was found in preparations of diabetic versus control hearts. Also azide failed to alter the measurements made, eliminating contamination by active mitochondrial components. The protein yield in the sarcoplasmic reticulum was similar between the two groups. Thus, although contamination can never be wholly excluded the evidence against it is strong in the present investigation. The possibility must be considered that the findings were not due to a diabetic effect but were caused directly by the effect of streptozotocin on the heart. A few studies were performed in female rats pretreated with 3-O methyl glucose, and SR preparations from the hearts of these animals in which diabetes had been partially blocked showed no depression in calcium binding and modestly lowered calcium uptake. Similarly, when decreased contractile protein function was observed in hearts of diabetic rats, that effect could be prevented by treatment with 3-O methyl glucose prior to the injection of streptozotocin [IO]. 3-O methyl glucose also prevents the delayed relaxation caused by streptozotocin diabetes when studies of myocardial mechanics are conducted [3]. Therefore, it appears that streptozotocin injection alone is not sufficient to cause deleterious effects on the heart, but that diabetes must develop for these abnormalities to become manifest.
308
S. Penpargkul
et al.
Streptozotocin induced diabetes results in a condition of caloric deprivation. The possibility must be considered that this, rather than the diabetic effect is responsible for the cardiac abnormalities. Although that possibility was not studied directly in the present investigation, a previous study in which control rats were subjected to caloric restriction to keep their heart and body weights the same as those of diabetic animals, did not reproduce the abnormalities in contractile function observed in diabetic animals [13]. Also caloric deprivation does not result in the marked depression in actomyosin ATPase activity that diabetes does [lo]. However, there was moderately delayed relaxation in hearts of food deprived animals, and it is possible that caloric restriction would have some effect on sarcoplasmic reticular function. Another factor that might be responsible for altered function of sarcoplasmic reticulum from hearts of diabetic animals might relate to the low circulating levels of thyroid hormone observed in these animals [3, S, 101. Decreased levels of circulating thyroid hormones are also observed in humans with diabetes [17]. The role of diminished circulating thyroid hormones in the streptozotocin diabetes syndrome and its effect on the heart were investigated previously by studying rats subjected to thyroidectomy and by comparing a group of diabetic animals and controls in which circulating thyroid hormone levels were similar [3, 101. The conclusions from those studies were that hypothyroidism could not explain the alterations either in contractile function or in contractile proteins observed in the presence of streptozotocin diabetes. The control data from this experiment suggest that sarcoplasmic reticular function may differ between hearts of male and female rats. Calcium uptake was about 30% lower in preparations from female controls than from male controls (Table 2). This difference has not been reported previously and requires further study. Depressed sarcoplasmic reticular activity has been observed in a number of physiologic and pathophysiologic states where contractile activity and relaxation are diminished. These include myocardial failure due to overload of the heart and cardiomyopathy in addition to hypothyroidism. It appears likely that this abnormality is at least partially responsible for the relaxation abnormality observed in the presence of diabetes [3, 131. Acknowledgements
We gratefully acknowledge the technical assistance of Mr Alwyn Murphy and Mr Alan Schwartz, the secretarial assistance of MS Christine Frawley, and for the editorial assistance of MS Carol Gundlach. This work was supported by U.S. Public Health Service Grants HL 20426 and HL 21482. Dr Fein is supported by U.S. Public Health Service Grant AM 00593.
REFERENCES 1.
2. 3. 4.
BERGMEYER, H. U., BERNT, E., SCHMIDT, F. & STORK, H. Determination with hexokinase In Methods of Enzymatic Analysis, Vol. 3, and glucose-6-phosphate dehydrogenase. H. U. Bergmeyer, Ed., pp. 1196-1201. London: Academic Press (1974). CROXTON, F. E. Elementary Statistics. New York: Dover (1959). FEIN, F. S:, KORNSTEIN, L. D., STROBECK, J. E., CAPASSO, J. M. & SONNENBLICK, E. H. Altered myocardial mechanics in diabetic rats. Circulation Research 47, 922-.933 (1980). FISKE, C. H. & SUBBAROW, Y. The calorimetric determination of phosphorus. Journal of Biological Chemistry 66, 375-400 (1925).
Sarcoplasmic 5. 6. 7.
8. 9. 10.
11. 12.
13. 14.
15.
16.
17. 18.
19.
Reticulum
in Diabetes
309
GANDA, 0. P., ROSSINI, A, A. & LIKE, A. A. Studies on streptozotocin diabetes. Diabetes 25, 595403 (1976). JOLIN, T. & GONZALES, C. Thyroid iodine metabolism in streptozotocin-diabetic rats. Acta Endocrinologica 88, 506-5 16 ( 1978). KATZ, A. M., REPKE, D. I., UPSHAW, J. E. & POLASCIK, M. A. Characterization of dog cardiac microsomes. Use of zonal centrifugation to fractionate fragmented sarcoplasmic reticulum (Na+ + K+)-activated ATPase and mitochondrial fragments. Biochimica et biophysics acta 205,473490 (1970). KANNEL, W. B., HJORTLAND, M. & CASTELLI, W. P. Role of diabetes in congestive heart failure: the Framingham study. American Journal of Cardiology 34, 29-34 (1974). LOWRY, 0. H., ROSEBROUGII, N. J., FARR, A. L. & RANDALL, R. J. Protein measurement with folin phenol reagents. Journal of Biological Chemistry 193, 265-275 (1951). MALHOTRA, A., PENPARGKUL, S., FEIN, F., SONNENBLICK, E. H. & SCHEUER, J. The effect of streptozotocin induced diabetes in rats on cardiac contractile proteins. Submitted for publication. PENPARGKUL, S. Effects of adenine nucleotides on calcium binding by rat heart sarcoplasmic reticulum. Cardiovascular Research 13, 243-253 (1979). PENPARGKUL, S., MALHOTRA, A., SCHAIBLE, T. & SCHEUER, J. Cardiac contractile proteins and sarcoplasmic reticulum in hearts of rats trained by running. Journal of ADplied Physiology : Respiratory Environmental and Exercise Pltysiolopy 48, 409-4 13 ( 1980). PENPARGKUL, S., SCHAIBLE, T., YIPINISOI, T. & SCHEUER, J. The effect of diabetes on performance and metabolism of rat hearts. Circulation Research 47, 9 1 l-92 1 ( 1980). REGAN, T. J., ETTINGER, P. O., KHAN, M. I., JESRANI, M. V., LYONS, M. M., OLDEWURTEL, H. A. & WEBER, M. Altered myocardia1 function and metabolism in chronic diabetes mellitus without ischemia in dogs. Circulation Research 35, 222-237 (1974). RECAN, T. J., LYONS, M. M., AHMED, S. S., LEVINSON, G. E., OLDEWURTEL, H. A., AHMED, M. R. & HAIDER, B. Evidence for cardiomyopathy in familial diabetes mellitus. Journal of Clinical Investigation 60, 885-899 (1977). RUBLER, S., DLUGASH, J., YUCEOGLU, Y. Z., KUMRAL, T., BRANWOOD, A. W. & GRISHMAN, A. New type of cardiomyopathy associated with diabetic glomerulosclerosis. American Journal of CardioloQ 30, 595-602 ( 1972). SAUNDERS, J., HALL, S. E. H. & SGNKSEN, P. H. Thyroid hormones in insulin requiring diabetes before and after treatment. Diabetologia 15, 29-32 (1978). SOLARO, R. J. & SHINER, J. S. Modulation of Ca2+ control of dog and rabbit cardiac myofibrils by Mg2+. Comparison with rabbit skeletal myofibrils. Circulation Research 39, 8-14 (1976). WHARTON, D. C. & TZAGOLOFF, A. Cytochome oxidase from beef heart mitochondria. In Methods in EnrymoloQ, Vol. 10, R. W. Estabrook & M. E. Pullman, Eds, pp. 245-250. New York: Academic Press (1967).
of Molecular
and Cellular
Depressed Somsong
Cardiology
Cardiac Penpargkul,
(1981)
13, 303-309
Sarcoplasmic from Diabetic Frederick and James
Reticular Rats
Fein, Edmund Scheuer*
Function H. Sonnenblick
Division
of Cardiology, Defiartment of Medicine, Montejore Hospital and Medical Center and Albert Einstein College of Medicine the Department of Physiology, Albert Einstein College of Medicine, Bronx, New York (Received 23 June 1980, accepted in revised form 19 November 1980) S. PENPARGKUL, F. FEIN, E. H. SONNENBLICK AND J. SCHEUER. Depressed Cardiac Sarcoplasmic Reticular Function from Diabetic Rats. Journal of Molecular and Cellular Cardiology (1981) 13, 303-309. Previous studies have demonstrated impaired contractile performance and delayed relaxation of hearts of diabetic rats. Male and female rats were made diabetic with intravenous streptozotocin and their hearts were studied 4 to 5 or 9 weeks later. Plasma glucose in female diabetics averaged 453 mg/lOO ml and in male diabetics 615 mg/lOO ml. Calcium uptake by isolated sarcoplasmic reticulum in the absence of oxalate were significantly lower in preparations from hearts of diabetic males and females than from controls. Rats pretreated with J-O-methyl glucose before streptozotocin also had normal calcium binding by SR in the absence of oxalate. Sarcoplasmic reticulum Mg2+ ATPase and Gas+-Mgsf (total) ATPase activities were significantly depressed in preparations from the diabetic animals. The results may partially explain the abnormalities in contraction and relaxation previously observed in hearts of diabetic animals. KEY
WORDS:
Calcium;
Streptozotocin;
ATPase;
Cardiac
relaxation;
Diabetes;
Sarcoplasmic
reticulum.
Introduction Previous investigations in experimental animals and in humans indicate that diabetes is associated with a specific cardiomyopathy [S, 14-161. Studies using papillary muscles [3] and isolated perfused hearts [23] from rats with streptozotocin induced diabetes demonstrate abnormalities in heart muscle function. The most prominent finding is a delay in the rate of relaxation. This defect persists in studies in which adequate oxygenation and energy generation appear to be assured, leading to the hypothesis that abnormalities exist in the energy utilizing mechanisms responsible for contraction and relaxation. Malhotra et al. [IO] examined contractile protein function in preparations of hearts of diabetic rats and found depressed Ca2+-activated myosin and actomyosin ATPase activities. These findings could explain abnormal cardiac contraction but not the slowing of relaxation. Since the calcium control by the sarcoplasmic reticulum plays an important role in modulating cardiac relaxation the following studies were conducted. Materials
and
Methods
Male Wistar rats weighing 180 to 200 g were made diabetic by injecting streptozotocin 50 mg/kg dissolved in 0.05 M citrate, pH 4.5 into the tail vein [23]. Control rats from the same initial group were injected with 0.05 M citrate only. Female * Reprint Bronx, New
requests to J. Scheuer, York 10467, U.S.A.
0022-2828/8
l/030303+
07 $02.00/O
Montefiore
Hospital 0
and Medical 1981 Academic
Center,
111 East 210th
Press Inc.
(London)
Street, Limited
304
S. Penpargkul
et al.
Wistars were injected with streptozotocin 60 mg/kg at initial weights of 170 to 200 g. All rats were allowed to ingest glucose in water for the next 24 h and were then fed normal Purina Rat Chow until they were killed. At weekly intervals, blood was drawn from the tail vein of control and diabetic animals for glucose determinations. Body weights were also recorded at weekly intervals to determine the growth rate. Details of blood analyses conducted on this model of diabetes have been outlined previously [IO, 131. Two groups (five to six rats each) of female rats were treated with 1.65 mmol of 3-O methyl glucose (Sigma) immediately before streptozotocin injection. This intervention prevents the diabetogenic effect of streptozotocin [J?, 51. Animals treated with 3-O methyl glucose were killed and studied at the same time as the diabetic female groups. Sarcoplasmic reticulum (SR) were isolated from pools of four to six ventricles as described by Penpargkul [II]. The homogenization was performed in 0.3 M sucrose and 10 mM imidazole (pH 7.4) using a Tekmar homogenizer. The final pellet was suspended in 50 mM KC1 and 10 mM imidazole, pH 6.8 (2 to 3 mg SR/ml). Calcium uptake was studied in the absence and presence of oxalate by the Mcllipore filtration technique. The reaction medium contained 120 mM KCl, 10 FM imidazole (pH 6.8), 5 mM MgCI,, 5 mM Tris-ATP, 7 mM phosphoenolpyruvate, 0.1 mg/ml pyruvate kinase, 5 mM sodium azide, 0.1 mM EGTA, 0.1 mM CaCl, and 0.15 mg SR protein/ml [12]. The study was performed at 25°C. The reaction was started by addition-of the?& For the assay of sarcoplasmic reticulum Mg a+-ATPase and Ca2+-Mg2+-ATPase activities, the reaction medium was similar to that used in calcium uptake. The total volume was 1 to 2 ml containing 0.05 mg SR protein/ml. For the Mg2+-ATPase determination the CaCI, was omitted from the reaction medium. The reaction was started by adding SR or ATP. At 1, 2, 4 and 10 min after starting the reaction, 0.2 to 0.5 ml was removed from the reaction tube and was added to 0.15 ml of 6% HCIO, to terminate the reaction. After centrifugation at 3000 g for 10 min at 2 to 4”C, an aliquot was taken for phosphate determination. Free calcium in the reaction medium was calculated by two different methods, as described by Katz et al. [7], and by Solar0 and Shiner [18] to be 5.4 pmol and 2.94 pm01 respectively. The cytochrome oxidase was determined by the method of Wharton and Tzagoloff [19]. Glucose was determined using an enzymatic method [I]. Inorganic phosphate was analyzed by the method of Fiske and SubbaRow [4]. The SR protein was measured by the method of Lowry et al. [9]. Analysis of sarcoplasmic reticulum preparations from control and diabetic animals were always performed simultaneously as paired samples. Statistical differences between mean values were evaluated by using analysis of variance for paired data [2].
Results
Streptozotocin induced diabetes causes plasma or serum glucose to increase rapidly within 24 h and to remain elevated until the time when rats were killed [IO, 121. Table 1 shows that in the current experiments plasma glucose was markedly elevated at the time of rats being killed in anesthetized animals. Table 1 also demonstrates that body weights, and heart weights were significantly less at the time of killing in
Sarcoplasmic
Reticulum
305
in Diabetes
diabetic animals than in controls, and in the present series of experiments, heart weight/body weight ratios were higher in the diabetic animals. Heart weight was depressed 30% in diabetic males and only 13 o/0 in diabetic females.
TABLE
1. Plasma
glucose,
heart
Body
weight
weight
and body Heart
(9)
Females
(4 to 5 weeks) control (9) diabetic 3-OMGt(l1)
Males
(9 weeks) control diabetic
weight
weight cd
rats
in diabetic
Plasma glucose (mg/lOO ml)
HW/BW b-+&d
(9)
230 f 186 f 238 f
4 7* 12
0.606 0.527 0.630
0.006 0.015* 0.012
2.63 2.84 2.65
f * f
0.003 0.006* 0.004
160 f 453 f 229 *
(9) (9)
447 f 263 f
8 14*
0.975 f 0.038 0.681 & 0.027*
2.19 2.60
f
0.005
170 615
Results are means i S.E. Numbers in parentheses are the number * P < 0.001 compared with control. t 3-O MG = 3-O methyl glucose treated
f j, f
relationships
of paired
f 0.005*
11 55* 20*
f 16 & 55*
studies.
rats.
Preparations of sarcoplasmic reticulum from hearts of females yielded SR protein of 0.73 & 0.07 mg/g in controls versus 0.83 & 0.08 in diabetics, and in males 0.69 & 0.05 mg/g in controls versus 0.63 f 0.06 in diabetics. The values between controls and diabetics were not significantly different. Cytochrome oxidase activity in preparations of sarcoplasmic reticulum indicated a similar degree of mitochondrial contamination (X = 7.1 f 1.3 min-r/mg control, vs. X = 5.4 f 0.4 diabetic). Table 2 gives the values for SR calcium uptake in hearts from female and male diabetic rats. Values for these functions were significantly lower in preparations of hearts of diabetics than from controls both in the presence and absence of oxalate. Rats treated with 3-O methyl glucose had heart and body weights similar to control rats and plasma glucose levels were elevated, but lower than found in diabetic animals. Calcium uptake by SR appeared to be similar to that found in control preparations when oxalate was absent. Calcium uptake was intermediate between control and diabetic where oxalate was present. Table 3 gives values of Mg2+ and Ca2+-Mg2+ activated ATPase from male and female diabetic rat hearts. There were marked depressions in Mg2+-ATPase of sarcoplasmic reticulum from hearts of male and female diabetic animals. SR isolated from hearts of normal female rats tended to have lower enzymatic activites than SR isolated from hearts of normal male rats. The calcium transport in the presence as well as absence of oxalate by the SR from female rat hearts was also lower than that of male rat hearts. Discussion
The present study shows that there are several abnormalities in isolated sarcoplasmic reticulum from hearts of rats made to be diabetic with streptozotocin. These include depressed calcium uptake and diminished Mg2+ and Ca2+-Mg2+-ATPase activities.
(oxalate
Female control Female diabetic Female 3-O MGT Male control Male diabetic
Casf-uptake (nmol/md
bmol/md Female control Female diabetic Female 3-O MGB Male control Male diabetic
(oxalate
Gas+-binding
Ca2+ uptake
2.
uptake
(8)
(8)
(8) (8) (2)
-
36.8 -& 1.9 27.8 f 1.2*
60.1 40.4 51.5 90.9 50.6
6.8 8.03
reticulum
f 7.8 & 9.8*
f f
hearts.
-
-
30 s
2.6
(7) (7)
present)
sarcoplasmic
4.38
32.4 23.6 41.6
f f
15s
by cardiac
(8) (8) (2)
omitted)
and
Results are mean & S.E. Numbers in parenthesis are the number of paired studies. * P < 0.001; t P < 0.01; $ P < 0.05. 7 3-O methyl glucose. n = two preparations of 5 to 6 pooled
TABLE
diabetic
3.5 5.93 3.9 2.5t
* f f
118 & 18 73 & 14s 66 166 f 13 lll-& 13*
44.1 67.5 64.6 49.4
f
1 min
59.4
from
animals
f f
* f
307 5 235 -j= 253 489 -& 346 &
79.8 56.5 98.5 97.4 65.3
4 min
37 39t
34 508
6.3 4.6t
6.9 5.9
649 521 620 940 714
184.0 109.0
129.2 77.5 136.7 18 11t
10 12t
f f
88 553
f 64 & 108
f f
f f
10 min
905 768 824 1142 925
z
-
f f
f f
15 min
134 889
67 148
Sarcoplasmic
TABLE
3. Sarcoplasmic
reticulum
Reticulum ATPase
activities
from
MgZ+-ATPase bWmd Females
1 min
control
diabetic 10 min control diabetic Males
1 min 10 min
control diabetic control diabetic
(5)
(5) (5) (5) (6) (6) (6) (6)
Results are mean + S.E. Numbers in parentheses are the number * = P < 0.001. t = P < 0.01.
3.50 2.18 22.8
f f
0.30 0.30t
i 14.6 f
1.0 1.61
4.38 f 2.50 f 28.9 f 15.8 f
0.30 0.34* 1.9 2.0*
of paired
307
in Diabetes hearts
of diabetic
rats
Ca2+ Mg2+-ATPase (wolbd 3.50 f 2.32 f 24.4 f 16.0 f 4.46 2.79 29.90 17.00
f f f f
0.30 0.237 1.60 1.6Ot 0.31 0.33* 2.2 2.0*
studies.
Studies of sarcoplasmic reticulum from cardiac muscle are always plagued by the possibility that unrecognized impurities of the preparations may result in artifactual conclusions. An inert contaminant that is contained in the preparations of diabetic hearts but not of the control hearts could explain depressions in enzymatic and calcium uptake characteristics of the SR from diabetic hearts. Such a contaminant would be expected to result in uniform differences for various measurements. In the present experiments the degree of depression of activities observed differed depending upon the measurement. For instance SR from male diabetic hearts showed 25,33 and 45% depressions in Gas+ uptake without oxalate, Ca2+ uptake with oxalate, and Mg2+-ATPase activity at 1 min respectively, suggesting that contamination with inert material was not responsible for these findings. The most likely active contaminant would be mitochondrial fragments. Measurements of cytochrome oxidase activity were made and no difference was found in preparations of diabetic versus control hearts. Also azide failed to alter the measurements made, eliminating contamination by active mitochondrial components. The protein yield in the sarcoplasmic reticulum was similar between the two groups. Thus, although contamination can never be wholly excluded the evidence against it is strong in the present investigation. The possibility must be considered that the findings were not due to a diabetic effect but were caused directly by the effect of streptozotocin on the heart. A few studies were performed in female rats pretreated with 3-O methyl glucose, and SR preparations from the hearts of these animals in which diabetes had been partially blocked showed no depression in calcium binding and modestly lowered calcium uptake. Similarly, when decreased contractile protein function was observed in hearts of diabetic rats, that effect could be prevented by treatment with 3-O methyl glucose prior to the injection of streptozotocin [IO]. 3-O methyl glucose also prevents the delayed relaxation caused by streptozotocin diabetes when studies of myocardial mechanics are conducted [3]. Therefore, it appears that streptozotocin injection alone is not sufficient to cause deleterious effects on the heart, but that diabetes must develop for these abnormalities to become manifest.
308
S. Penpargkul
et al.
Streptozotocin induced diabetes results in a condition of caloric deprivation. The possibility must be considered that this, rather than the diabetic effect is responsible for the cardiac abnormalities. Although that possibility was not studied directly in the present investigation, a previous study in which control rats were subjected to caloric restriction to keep their heart and body weights the same as those of diabetic animals, did not reproduce the abnormalities in contractile function observed in diabetic animals [13]. Also caloric deprivation does not result in the marked depression in actomyosin ATPase activity that diabetes does [lo]. However, there was moderately delayed relaxation in hearts of food deprived animals, and it is possible that caloric restriction would have some effect on sarcoplasmic reticular function. Another factor that might be responsible for altered function of sarcoplasmic reticulum from hearts of diabetic animals might relate to the low circulating levels of thyroid hormone observed in these animals [3, S, 101. Decreased levels of circulating thyroid hormones are also observed in humans with diabetes [17]. The role of diminished circulating thyroid hormones in the streptozotocin diabetes syndrome and its effect on the heart were investigated previously by studying rats subjected to thyroidectomy and by comparing a group of diabetic animals and controls in which circulating thyroid hormone levels were similar [3, 101. The conclusions from those studies were that hypothyroidism could not explain the alterations either in contractile function or in contractile proteins observed in the presence of streptozotocin diabetes. The control data from this experiment suggest that sarcoplasmic reticular function may differ between hearts of male and female rats. Calcium uptake was about 30% lower in preparations from female controls than from male controls (Table 2). This difference has not been reported previously and requires further study. Depressed sarcoplasmic reticular activity has been observed in a number of physiologic and pathophysiologic states where contractile activity and relaxation are diminished. These include myocardial failure due to overload of the heart and cardiomyopathy in addition to hypothyroidism. It appears likely that this abnormality is at least partially responsible for the relaxation abnormality observed in the presence of diabetes [3, 131. Acknowledgements
We gratefully acknowledge the technical assistance of Mr Alwyn Murphy and Mr Alan Schwartz, the secretarial assistance of MS Christine Frawley, and for the editorial assistance of MS Carol Gundlach. This work was supported by U.S. Public Health Service Grants HL 20426 and HL 21482. Dr Fein is supported by U.S. Public Health Service Grant AM 00593.
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