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Left ventricular function after chronic insulin treatment in diabetic and normal rats
J Mol
Cell
Left
Thomas
Cardiol
15, 445-458
Ventricular
(1983)
Function in Diabetic
F. Schaible,
Ashwani
After Chronic and Normal
Malhotra,
William
Insulin Rats
Treatment
A. Bauman
and James
Scheuer
Departments of Medicine and Physiology and Biophysics. Albert Einstein College of Medicine, and The Division of Cardiology and Department of Clinical Sciences, MonteJiore Hospital and Medical Center, Bronx, New York 10467, USA. (Received 3 August 1982, accepted in revised form 17 January
1983)
T. F. SCHAIBLE, A. MALHOTRA, W. A. BAUMAN AND J. SCHEUER. Left Ventricular Function After Chronic Insulin Treatment in Diabetic and Normal Rats. Journal of Molecular and Cellular Cardiology (1983) 15, 445458. Previous reports have documented a cardiomyopathy in rats resulting from streptozotocininduced diabetes. In order to determine the reversibility of streptozotocin-induced cardiomyopathy to insulin therapy, hearts from rats made diabetic by streptozotocin for 6 weeks and then treated with insulin for 3 weeks were compared with untreated diabetic rats and control rats not injected with streptozotocin. When perfused in an isolated working heart apparatus with 5.5 mM glucose as substrate, hearts from untreated diabetic rats when compared to hearts from either streptozotocin-injected rats treated with insulin or control rats showed significant depressions in peak left ventricular pressure, maximal positive and negative dP/dt, oxygen extraction, lactate production and effluent lactate; pyruvate ratio. Ca2+-actomyosin ATPase was also depressed in untreated diabetics. As left atria1 pressure was raised in untreated diabetic rats, a decline in cardiac output was observed, whereas in insulin-treated or control groups there was no such negative response. Indices of cardiac performance were significantly greater in insulin-treated rats when compared to control rats suggesting overcorrection with insulin therapy. To explore whether insulin treatment may have a beneficial effect on the myocardium control rats were made hyperinsulinemic for 6 to 7 weeks. Shorter isovolumic relaxation times and elevated values for Ca2+actomyosin ATPase were observed in hearts from hyperinsulinemic animals when compared to hearts from control animals. These results demonstrate complete reversibility of streptozotocin-induced cardiomyopathy and confirm that this condition is due to insulin deficiency and not to a primary cardiotoxic effect of streptozotocin. KEY WORDS: insulinemia.
Diabetes
mellitus;
Ventricular
function;
Introduction Insulin deficient diabetes has been associated with a specific cardiomyopathy in humans [23, 251 and experimental animals [IO, 14, 19, 221. Most animal studies have relied on chemical substances as diabetogenic agents which purportedly selectively destroy the beta cells of the pancreas, the primary source of insulin synthesis. Previous studies have seldom incorporated a diabetic group treated with insulin in the experimental design so that untoward effects of the agent may be assessed. In some studies that have included such a group, only partial reversal of diabetesrelated abnormalities has occurred with insulin treatment [IS, 17, 241. OOZZ-2828/83/070445+ M.C.C.
14 $03.00/O
Actomyosin
ATPase;
Insulin
therapy;
Hyper-
A previous report from this laboratory [19] characterized a cardiomyopathy in rats made diabetic for eight weeks by a single injection of streptozotocin. Isolated working hearts from streptozotocin-injected animals showed depressed cardiac contractile function and impaired relaxation, despite normal coronary flows and myocardial oxygen consumptions. In order to determine the extent to which the cardiac abnormalities found in streptozotocin diabetes could be reversed, the present study was designed using a streptozotocin injected group that was subsequently treated daily with insulin. Our results demonstrated that the cardiomyopathy associated with streptozotocin-induced diabetes could 0
1983 Academic
Press Inc.
(London)
Limited u
446
T. F. Schaible
be reversed by insulin therapy. In addition, hearts from streptozotocin diabetic rats that were treated with insulin demonstrated better performance than hearts from control animals that were not injected with streptozotocin. Therefore, a second study was designed to determine whether chronic insulin treatment would have a beneficial effect on the myocardium.
Methods
Experimental
groups
Experiment 1: The e$ects of insulin treatment on streptozotoci’n-induced diabetic cardiomyopathy. Male Wistar rats initially weighing 180 to 200 g were fasted overnight and then were made diabetic the following morning by injecting streptozotocin, 50 mg/kg, dissolved in 0.05 M citrate, pH 4.5, into the tail vein. Control rats from the same initial group were not injected. All animals were fed Purina Rat Chow and watered ad libitum for the duration of the study. Six weeks after injection the diabetic group was subdivided into a group that received insulin treatment and a group that did not receive treatment. Animals were placed in the insulin-treated and non-treated diabetic groups according to similar blood glucose levels and body weights. Protamine zinc insulin (Lilly) was injected subcutaneously at approximately 4 p.m. every day. Blood glucose and body weight were measured in all of the insulin-treated animals at 3 to 4 day intervals and in some of the non-treated and control animals at weekly intervals. Blood samples were drawn at 9 to 10 a.m. from the tail vein of conscious restrained rats. Rats in the insulin-treated group received treatment (3 to 6 U/day) for 18 to 30 days at which time they were sacrificed and their hearts were studied along with hearts from rats in the non-treated diabetic and control groups. Rats in the insulin-treated group did not receive insulin for 18 h prior to the time their hearts were studied. Experiment 2: The e$ect of hyperinsulinemia on cardiac pe$rmance. Hyperinsulinemia was produced in two groups of rats. The first group of rats (CI,) weighed approximately 400 g when daily insulin injections were
et al.
started. The second group of rats (CI,) weighed approximately 200 g when treatment began. Rats in both groups were injected initially with 4 U Protamine zinc insulin daily. This dosage was progressively increased to 7 U daily over the course of the study. Both hyperinsulinemic groups received a 7.5:/, (w/v) dextrose solution as drinking water to avoid deaths from hypoglycemia. Treatment lasted for 40 to 50 days at which time the hearts from treated rats were studied and compared to hearts from age-matched untreated rats (C, and C,) . During the course of the study blood samples were drawn from the tail vein to determine glucose and insulin levels. Details for the timing of insulin injection and blood collection were the same as in Experiment 1. Heart perfusions: Full descriptions of the isolated working rat heart apparatus have been published previously [2, 26, 271. The perfusate was a modified Krebs-Henseleit buffer at 37°C gassed with a 95:/, 0, and 5!/, CO, mixture and containing 5.5 mM glucose, and 2.0 mM calcium with 0.5 mM EDTA yielding 1.5 mM free calcium. The perfusate was not recirculated. Hearts in Experiment 1 were perfused without insulin while hearts in Experiment 2 were perfused with a buffer containing 0.01 U/ml regular insulin (Lilly). Insulin was included in the buffer in Experiment 2 to avoid the possibility that a higher residual insulin concentration in hearts from hyperinsulinemic animals would contribute to improved function. Left ventricular pressure was measured through a 2.5-cm polyethylene (PE-60) catheter inserted through the apex of the heart and attached to a Statham P23dB strain gauge pressure transducer. A second catheter was placed through the left ventricular apex for dye injection. Aortic pressure was measured from a sidearm on the aortic cannula approximately 7 to 8 mm above the aortic valve. The frequency responses of the pressure measuring systems were flat f lo:/, to 30 Hz. Instantaneous aortic ilow was measured from a cannulating 2.5 mm I.D. flow probe (Statham-Gould) inserted in the aortic outflow tubing. Coronary flow was measured directly as right heart outflow, and cardiac output was measured as aortic flow plus coronary flow. Dye concentrations in the aorta were
Reversal
of Diabetic
measured by a densitometer system placed into the aortic cannula. All hearts were paced from the right atrium at a rate of 340 beats/ min. Oxygen tension was measured in the perfusate from the left atria1 reservoir and from a pulmonary arterial catheter. Arteriovenous differences in oxygen tension were converted to oxygen consumption by multiplying the arteriovenous oxygen tension by coronary flow and the appropriate Bunsen coefficient 1321 and dividing by left ventricular dry weight. Samples were taken from the atria1 reservoir and right ventricular outflow for analysis of lactate and pyruvate. Whenever possible, one heart from each group within an experiment was perfused on a given day. Cardiac function was assessed as a function of preload, which was varied by changing the height of the reservoir filling the left atrium (left atria1 pressure). The height of the aortic column was 80 cm at all times. After a 10 to 15 min period of retrograde during which catheters for left perfusion, ventricular pressure and dye injection were positioned, antegrade perfusion was begun by unclamping the left atria1 cannula and the heart was allowed to equilibrate for 15 min at a left atria1 pressure of 10 cm H,O. After an initial recording of data at 10 cm HZ0 the filling pressure was lowered to 5 cm H,O, then raised in sequence to 10, 15, and 20 cm H,O and finally lowered to 10 cm H,O, all for periods of 7.5 min. After 5 min at each filling pressure, records were made of dynamics, multiple dye dilution curves were recorded and the coronary flow and cardiac output were measured over a I-min period. All analog data (dye concentration, aortic flow and left ventricular and aortic pressure) were stored on magnetic tape for later analysis on a digital computer. End-diastolic volumes were estimated from the dye-dilution curves. A full description of this method and the validation of its accuracy for measuring ventricular volumes have been published previously [2]. End-diastolic volume was calculated by -dividing the directly measured stroke volume (cardiac output divided by heart rate) by the ejection fraction determined from the multiple dye curves. Flow and pressure analog data were M.C.C.
44.7
Cardiomyopathy
digitized at 660 samples/s and the measurements of cardiac dynamics were determined as previously described [2, 271. Geometric calculations were based on the assumption that the left ventricule has a spherical geometry with a constant wall thickness as described presriously [27]. By integrating aortic flow during ejection, the ejected volume could be subtracted from the enddiastolic volume, yielding instantaneous left ventricular volume. This permitted calculation of the velocity of circumferential fiber shortening ( V,$, total circumferential fiber shortening, and fractional shortening at th.e midwall. Instantaneous wall stress was estimated from the La Place equation [27] and The isovolumic reexpressed in g/cm. laxation time was calculated as the period from the onset of the dicrotic notch (aortic valve closure) until the left ventricular pressure relaxed to the left atria1 pressure (mitral valve opening). At the end of each experiment the atria and great vessels were dissected free and the right ventricular free wall was removed. The left ventricle (including the septum) wals weighed to determine wet weight. A small piece of the left ventricle (approximately 0.2 g wet weight) was dried to constant weight in an oven to calculate left ventricu1a.r dry weight. The remainder of the left ventricule was retained for analysis of actomyosin ATPase. Actomyosin A TPase All hearts were stored at -80°C in 5OqJ, glycerol containing 50 mM KC1 and 10 rnlti KPO,(pH 7.0) prior to preparing extracts. Hearts of experimental animals were always extracted and analyzed simultaneously with hearts of controls using the same reagents and incubation conditions. Actomyosin was used because this permits the evaluation of muscle from individual hearts, rather than necessitating the pooling of several hearts which is required when pure myosin is studied. The measurement of actomyosin ATPase activity in high ionic strength (0.3 M KCl) is similar to measuring pure myosin, and in severaL studies from our laboratory there has been excellent correspondence without exception, between this measurement, calcium activated
448
T. F. Schaible
ATPase and actin activated myosin ATPase [12, 133. The methods of preparing and analyzing cardiac actomyosin from individual hearts have been detailed previously [5]. Results are expressed as pmol Pi liberated per min per mg protein at 30°C. Chemical analyses Lactate and pyruvate were determined in perfusate samples by the methods of Hohorst [11] and Segal [30] respectively. Glucose in 37; perchloric acid extracts from blood was determined by a hexokinase : glucose-6phosphate dehydrogenase method [I]. The concentration of insulin in the plasma samples was determined by radioimmunoassay using two guinea-pig antisera with different sensitivities and species-specificities, according to methods previously described [3, 4, 331. The assay system employing GP 11-8-14 had a minimal sensitivity for the detection of beef insulin of 10 pg/ml incubation mixture. Rat insulin crossreacted tenfold more weakly in this system. Plasma insulin in the beef-insulin treated animals was assayed with this antiserum employing 12sI-beef insulin as the tracer and Lilly beef insulin as standard. Since endogenous rat insulin would be suppressed by insulin treatment, none would be measured in this system. The assay system employing 1251-pork insulin as the tracer and GP N-3-13 was capable of detecting 3 pg Lilly rat insulin/ml and was used for determination of endogenous rat insulin in non-insulin-treated animals. Each plasma sample was assayed at a dilution of plasma 1: 20 or greater. There were no detectable anti-insulin antibodies at these plasma dilutions. Tibia1 lengths When the animals were sacrificed, both tibia bones were dissected out. These were dried in an oven and then measured to the nearest 0.1 mm under a magnifying glass. The length was taken as the distance from the condyles to the tip of the medial malleolus [34]. The dry left ventricular weight to dry tibia1 length ratio was compared to the dry left ventricular weight to body weight ratio for each group of animals in Experiment 1.
et al.
Statistical
anaCysis
Results at any given filling pressure for the three groups of animals in Experiment 1 were first submitted to a one way analysis of variance [35] to test for any group differences. If a significant F value was obtained (P < 0.05), differences between any two groups were tested by the Neuman-Keuls multiple comparison test [35]. In Experiment 2 where only two groups were compared the unpaired student t test was employed [35]. Significant differences at the P < 0.05 and P < 0.001 level are reported.
Results
Experiment
1
Figure 1 shows the time course changes in blood glucose (upper panel) and body weight (lower panel) for the control, untreated diabetic rats and streptozotocin-injected rats treated with insulin groups. Insulin dosages for the treatment schedule are also shown. After 5 days using 5 to 6 U/day protamine zinc insulin, glucose values in the insulintreated group declined to values in the control group and remained at this level using dosages of 3 to 4 U/day. Values for the untreated diabetic group remained above 400 mg%. Body weights in the insulintreated group increased substantially once insulin therapy was initiated. Table 1 shows some of the gross morphological measurements in the three groups. For measurements of dry heart weight, body weight and dry tibia1 length, the three groups were statistically autonomous and values for insulin-treated were intermediate between values for control and untreated diabetic groups. For the dry heart weight to body weight ratio, values were similar between untreated diabetic and insulin treated groups and both of these were greater than the value for control. Conversely, for the dry heart weight to tibia1 length ratio, values were similar between control and insulin-treated and both of these were greater than the value for untreated diabetic group. Therefore, heart size relative to body size was reversed in insulin treated diabetes only when expressed as the dry heart weight to tibia1 length ratio.
Reversal
of Diabetic
Cardiomyopathy
44:9
&/450
-
400
i/-
-
s i .P $ p” 4
FIGURE 1. Values for blood glucose and body weight in diabetic, diabetic treated with insulin and control animals during the course of insulin treatment. The diabetic and treated diabetic groups had been diabetic 6 weeks prior to the beginning of insulin treatment. Daily insulin dosages during the treatment schedule are shown at the bottom. Results for body weight for all groups and for glucose in the treated diabetic group represent the mean i SAM. for 10 to 17 animals. Glucose values for the diabetic group and control group, represent the average of values for 3 to 4 animals. O----a, control; A’- - -[l, diabetic; A- - -A, treated diabetic.
350
-
300
-
250
-
i,-“*
O-
_A
.,i-----i’ I- -
,,I-
**i.---
,’ _ -,/_____-- -----&+
_________ A ___. - -----
I
I
0
4
I 8
I I2
/-- i
_/ -
a
-----------
I 16
I 20
24
Days k-6-4-5
a-3-4 Insulin
TABLE
1. Results
for
body
weight,
dose
!U:
heart
weight
and
tibia1
length
DHW/BW n Experiment
Cd
DHW
(m.d
DHW/DTL DTL
bgld
11 11 11
Experiment
491 f 12” 296 f 7 375 & ll”,d
233 & 5b 17064 210 -& 6b+
0.475 0.575 0.562
f f f
0.009b 0.010 0.008d
4.18 3.76 3.89
f f f
0.030 0.030 0.027-
55.8 44.8 54.0
f l.lb & 0.9 & 1.3b
2
G
7
498
f
20
241 &
15
0.482
f
0.018
7
495
f
11
242 f
4
0.490
*
0.014
4
CI,
(mdcm)
1
CI, Group G
(cm)
1
c D DI Group
BW
-
-
2 7
390
-& 9
184 f
7
404
f
201 & 3”
5
0.472
& 0.007
-
0.495
f
-
0.007”
Values arc mean & S.E. BW, body weight; DHW, dry heart weight; DTL, dry tibia1 length. For Experiment 1: &P < 0.05, bP < 0.001, for C v. D or DI v. D; c,P < 0.05, *P < 0.001 for DI v. C. For Experiment 2: a P < 0.05 for C, v. CI,. C, control; D, untreated diabetic; CI, insulin-treated diabetic.
-
450
T. F. Schaible
Measurements of cardiac output and stroke work (both per g dry left ventricle) are shown in the top panels of Figure 2. Hearts from the insulin-treated group demonstrated a significantly higher cardiac output than both untreated diabetic and control groups at the highest left atria1 pressure. Stroke work for the insulin-treated group was significantly greater than control and untreated diabetic groups at 15 and 20 cm atria1 pressure, but, in addition, values for control were greater than values for the untreated diabetic group at the highest left atria1 pressure. As atria1 pressure was increased from 15 to 20 cm H,O values for both cardiac output and stroke work decreased in the untreated dia-
et al.
betic group (P < O-005), remained the same in controls (P > 0.05) and increased in the insulin-treated group (P < 0.005). Values for peak left ventricular systolic pressure and maximum positive dP/dt are shown in the bottom panels of Figure 2. Values for both of these functions were significantly greater in the control and insulin-treated group compared to untreated diabetics at all left atria1 pressures. At some atria1 pressures, peak left ventricular systolic pressure was greater in the insulin-treated group than control. Figure 3 shows data for two measurements of ventricular relaxation, namely the maximum rate of left ventricular pressure decline (max - dP/dt) and the isovolumic relaxa-
1.25
-
1.00
-
$j
f
Left
atrial
filling
pressure
(cm
H:! 0)
FIGURE 2. Measurements of pump performance as a function of left atria1 filling pressure for the groups in experiment 1. Cardiac output and stroke work are shown in the top panels and are normalized for dry left ventricular weight. Results represent the mean & 1 S.E. In addition, paired t statistics showed a significant increase (P < 0.05) in cardiac output and stroke work going from 15 to 20 cm Hz0 atria1 pressure for hearts from treated diabetic animals, no change (P > 0.05) in hearts from control animals, and a significant decrease (P < 0.05) in hearts from diabetic animals. Pressure related measurements from hearts in Experiment 1 are shown in the bottom panels. PLVSP = peak left ventricular systolic pressure; Max + dP/dt = maximum rate of left ventricular pressure rise. e--e, control (11) ; ,& - -A, diabetic (11) ; ); - -A, treated diabetic (11). < 0.001 for control v. untreated Numbers in parentheses indicate the number of preparations. *P < 0.05, . **P diabetic or insulin-treated diabetic v. untreated diabetic; +P < 0.05 for insulin-treated diabetic v. control.
Reversal
of Diabetic
I IO Left
atrial
FIGURE 3. Measurements of left ventricular maximum rate of left ventricular pressure decline; the same as in Figure 2.
filling
pressure
1”
3 L$
ij
E ; P :: -I5
2.5 2.0
4 01‘ IO’
I 5
I IO
I I5
I 20
atria1
I 20
I lo-
(cm l-l, 0)
All
1. Max - dP/dt == other symbols are
200 175 150
a 4
125
O-
I IO Left
I 15
V,f
::
I -I
I IO
similar in untreated and diabetic control groups but values at high left atria1 pressure were significantly greater in insulin-treated compared to either untreated diabetic or control groups. Fractional shortening calculated at the midwall showed patterns of differences that were similar to those observed for ejection fraction. Maximum was greater in insulin-treated than both control and untreated diabetic groups at all left
“6 3.0
I 5
relaxation in hearts from Experiment Isov. relax = isovolumic relaxation.
tion period. Values for control and insulintreated groups were similar and significantly different from values for the untreated diabetic group. The isovolumic relaxation period was nearly twice as long in the untreated diabetic group as compared to that observed in insulin-treated or control groups. Figure 4 shows values for ejection fraction and derived measurements for shortening, force and velocity. Ejection fraction was
.-:
451
Cardiomyopathy
filling
FIGURE 4. Measurements offorce, velocity and shortening velocity of circumferential fiber shortening. All other symbols
pressure
(cm Hp 0)
in hearts from Experiment are the same as in Figure
1. Max 2.
V,f = maximum
452
T. F. Schaible
atria1 pressures. However, maximum wall stress was similar in insulin-treated and control and greater than in the untreated diabetic group at the higher filling pressures. Figure 5 shows that end-diastolic pressure was Iower in the untreated diabetic group than either control or insulin-treated groups at all but the lowest left atria1 pressure. Similar relationships amongst the three
et al.
groups were observed for end-diastolic wall stress, although these data are not shown. Figure 5 also shows that end-diastolic volume (per g dry left ventricle) was not different amongst the three groups. Data describing oxygen delivery and utilization are shown in Table 2. Hearts from the untreated diabetic group demonstrated greater coronary flows per g at all left atria1
6-
3-
o-
I
I
I
I
I
I
IO
5
IO
15
20
IO Left
FIGURE 5. End-diastolic in hearts from Experiment as in Figure 2.
TABLE
2. Values for Experiment
coronary 1
oxygen
flow,
Left
Oxygen
C D DI Myocardial C D DI
Jaw
(ml/g/min) 87 f 3” 98 zt 4 84 f 2”
extraction 0.642 0.561 0.669
filling
pressure
extraction
atria1 pressure 10
92 f 3” 104 + 3 90 f 2a
I 5
’
IO (cm
pressure (EDP) and end-diastolic volume 1. EDV is expressed per g dry left ventricular
5 Coronary C D DI
atria1
01
I IO
I 15
I 20
L I(
Hz 0)
as functions (EDV) weight. All other
and
oxygen
of left atria1 pressure symbols are the same
consumption
for
hearts
in
(cm H,O) 15
20
92 5 38 104 f 4 90 f 2”
92 + 3” 106 f 3 91 * 3a
(ml/ml) + 0.014s + 0.017 * 0.011”
oxygen consumption 1.10 f 0.03 1.09 & 0.02 1.12 * 0.02
0.667 0.593 0.717
f 0.012b -& 0.014 + 0.Ollb.c
(mllglmin) 1.21 & 0.04 1.22 * 0.03 1.29 * 0.03
0.684 0.598 0.748
& 0.013b 5 0.015 & 0.012b.c
1.24 f 1.24 j, 1.35 *
0.03 0.03 0.04
Values represent the mean & 1 S.E. of 11 hearts. Coronary flow and oxygen g dry weight. BP < 0.05; bP < 0.001 C v. D or DI v. D.; CP < 0.05 DI v. C. Abbreviations: see Table 1.
0.697 0.591 0.748
& 0.012” & 0.017 & 0.017bsc
1.25 f 1.24 + 1.36 * consumption
0.03 0.03 0.04
are expressed
per
Reversal
of Diabetic
pressures when compared to either insulintreated or control groups. Oxygen extraction was less in the untreated diabetic group compared to either insulin-treated or control groups and it was less in control than in insulin-treated groups. Myocardial oxygen consumption was similar in all three groups but there was a trend towards higher values in insulin treated compared to either control or untreated diabetic groups. Values for lactate and pyruvate production are shown in Figure 6. Hearts from the untreated diabetic group produced larger amounts of pyruvate and lesser amounts of lactate when compared to either control or insulin-treated groups. In addition, lactate production and the sum of lactate and pyruvate production were greater in insulintreated than control groups at the higher left atria1 pressures. The ratio of lactate to pyruvate production was substantially less in
Cardiomyopathy
the untreated diabetic group when compared to either control or insulin-treated groups. Table 3 shows values for Ca2+ stimulated actomyosin ATPase for the three groups in Experiment 1. ATPase activity was approximately 50°j, depressed for the untreated diabetic group compared to either control or insulin-treated groups. There was a significant 17:/:, elevation in enzyme activity in the insulin-treated group when compared to control.
Experiment2 Blood glucose and plasma insulin levels obtained during the second and fourth weeks of insulin treatment are shown for treated and untreated animals from Group 2 in Table 4. Plasma insulin levels were four to six times greater in insulin-treated animals at both time points. Blood glucose levels were not different between C, and CI, at the
1
T+
8-
4513
I
I
I
I
I
** l ---____ I **
12 (u
6-
I ** &./I** z /-I 0 * * L6-r* ?----*5
1
5
IO
15
20
IO Left
All A-
30
0, IO
I** I**
Y5 5 9-
-I
atria1
filling
I
*n
pressure
4
~ ______
a _--- ---A-------4
I IO
I 5
I IO
I 15
4 1 20
I IO
(cm H,O)
FIGURE 6. Values for lactate and pyruvate production, their sum and their ratio for hearts in Experiment values are expressed per g dry left ventricular weight. O---e, control (9); A- --A, diabetic - -A, treated diabetic (10).
1. (9);
454 TABLE
T. F. Schaible
3. Gas+-Actomyosin
ATPase
et al.
values for groups in Experiments
n
1 and 2
Ca2+-Actomyosin ATPase (pm01 Pi/mg/min)
Exfieriment 1 C D DI
7 9 6
E@eriment 2 Group 1 Cl CI,
7 10
0.576 & 0.017 0.678 & 0.013”
Groufi 2 G CIZ
8 8
0.539 & 0.015 0.690 i 0.04Za
0.566 & 0.017b 0.281 5 0.017 0.680 & 0.028bsc
Values are mean & S.E.; n = number of hearts. For Experiment 1: b&’ < 0.001 for C v. D or DI v. D; CP < 0.05 for DI v. C. For Experiment 2: BP < 0.005; bp < 0.001 for C1 v. CI,, or C, v. CI,. Abbreviations: see Table 1.
TABLE
4. Blood glucose and plasma insulin levels from control and insulin treated animals in Group 2 from Experiment 2 4 weeks
2 weeks G C* c12
106 h 4 (5) 82 i 11 (‘5)
I
G
19 & 4 (5) 87 & 9” (‘1)
126 f 7 (5) 58 f 6” (13)
I 141 1 (4) 97 * 14” (13)
Samples were obtained during the second and fourth week of insulin treatment. Values are means f 1 S.E. Number of observations are shown in parentheses. G = blood glucose (mg/lOO ml blood); I = plasma insulin (pU/ml plasma). =P < 0.005; bP < 0.001 CI, v. Cz. Abbreviations : see Table 1.
two-weeksamplingpoint butwere significantly lower in CI, at the 4 week sampling point. Table 1 shows data for heart and body weights from insulin-treated and control animals. Heart and body weights were similar between C, and CI, in Group 1. However, in Group 2 where the treatment was started at an earlier age, animals from the treated group (CI,) had mildly elevated heart weights and heart weight to body weight ratio when compared to the untreated group (Cs) .
Data for performance of isolated hearts are shown in Table 5. All flow, volume and contractile measurements were similar in comparing treated and untreated groups in either Group 1 or Group 2. The only consistent differences observed involved measurements of cardiac relaxation. Thus, in Group 1, CI, demonstrated a significantly greater rate in the decline of left ventricular pressure (max - dP/dt) when compared to C, while for both Group I and 2, insulin-treated
Reversal TABLE
5. Dynamic
performance
of Diabetic
in isolated
hearts Group
455
Cardiomyopathy from
Experiment
2 Group
1
2
LAP (cm H@) Coronary (mlidmin)
flow
15 20
Cardiac (ml/g/min)
Output
15 20
87 f 87 f
2 2
517 & 25 525 & 2.5
673 f 676 *
23 24
639 5 22 647 & 25
8.1 * 0.4 9.6 & 0.9
8.7 f 10.5 i
0.6 0.8
8.2 & 0.4 9.0 & 0.5
8.3 + 0.3 8.7 f 0.1
Peak LV Pressure (mmHg)
15 20
104 f 103 *
107 f 107 f
2 2
108+ I07f
113 & 3 112 f 3
Ejection
15 20
0.51 0.52
f 0.03 3 0.04
0.58 0.57
f 0.02 & 0.01
0.63 0.62
3 0.04 f 0.04
0.60 0.60
Max-dP/dt
15
(mm%/s)
20
2720 2610
f 103 + 92
3060 2910
f 136 & 103a
2790 2680
* 128 & 132
2530 2420
Iso”
15 20
17.2 & 1.2 17.6 & 1.7
& 0.03 * 0.08
12.7 f 1.0a 12.6 & l.la
3.18 3.23
rt: 0.16 f 0.14
1 I
19.5 + 0.9 20.3 & 1.5
3.22 3.30
f i
4 3
15 20
2 2
2.69 2.82
103 f 105 f
End-diastolic Pressure (mmHg)
Relax
0.18 0.25
22 24
3 3
15 20
Time (ms)
f *
530 f 551 *
107 f 106f
End-diastolic Volume (ml/g)
fraction
3.02 3.04
83 & 3 86 f 3
0.23 0.32
i 0.03 & 0.04 * *
72 52
14.2 & O.Yb 14.4 * 0.7b -
Values represent the mean & 1 SE. of 7 hearts in each group. LV. LAP = left atria1 filling pressure. “I’ < 0.05; bP < 0.005 for C1 v. CI, or C, v. CI,. Abreviations: see Table 1.
groups had significantly shorter insvolumic relaxation times when compared to their respective untreated group. Table 3 summarizes values for Ca2+stimulated actomyosin ATPase for the groups in Experiment 2. Significantly higher values for enzyme activity in treated animals were observed for the comparisons C, v. CI, and C, v. CI,. These differences amounted to 18% and 28:/, respectively.
Discussion
In the present study, treatment of the streptozotocin diabetic animals with insulin completely reversed all mechanical and metabolic abnormalities observed in isolated working hearts from streptozotocin-diabetic animals. These findings in addition to the finding that depressed levels of cardiac actomyosin ATPase were also reversed in insulin therapy, provide evidence that the streptozotocin effect was not one of direct
Flow
and volume
data
are expressed
per g dlry
toxicity on the heart but related to a reversible diabetic state. The present findin.gs are directionally similar to those reported by Fein et al. [9] who found partial reversal of mechanical abnormalities in papillary muscles after 10 days of insulin treatment in female diabetic rats and complete reversal after 28 days of insulin treatment. This wa.s accompanied by normalization of contractile protein enzyme activity in the same time frame. In a recent study by Murphy et al., [1S] male rats made diabetic with streptozotocin and subsequently treated with 2 U protamine zinc insulin daily for 4 weeks, did not fully recover maximal heart rate response or performance time in a graded treadmill exercise test. The discrepancy between these findings and our own may be due to the possibility that factors that control heart rate may not be completely reversed by insulin therapy in streptozotocin-diabetic rats. The myocardial interstitial fibrosis that occurs in alloxan diabetic dogs is not affected
456
T. F. Schaible
by insulin treatment [24], but there is no fibrosis in streptozotocin diabetic rats [8, 1.51. Thus, diabetic cardiomyopathies involving interstitial fibrotic abnormalities may not be as amenable to reversal by insulin therapy as the diabetic cardiomyopathy in this study which primarily involves contractile dysfunction. Particularly notable in the present studies is evidence that treatment of diabetic animals with insulin resulted in cardiac performance that was greater than observed in control hearts. Values for cardiac output, stroke work, peak pressure development, ejection fraction, fractional shortening and maximal were significantly greater in insulinVCf treated than in control groups. In addition, values for cardiac output and stroke work significantly increased in insulin-treated when the left atria1 pressure was raised from 15 to 20 cm H,O while values in control remained the same. These findings may represents true improvements in contractile performance end-diastolic since end-diastolic pressure, volume and maximal wall stress were similar in insulin-treated and control groups. However, two factors related to our methodology may contribute, in part, to these differences. The first of these involves the differences in heart mass between insulin-treated and control groups, which was 10% less in insulin-treated than control. We have previously shown that small hearts demonstrate higher values for cardiac output per g when compared to large hearts from older animals [19]. However, force, velocity and shortening measures of contractile performance are not as dependent on heart mass as normalized cardiac output. Our studies [29) would predict no differences in peak pressure development, a 4:(, increase in ejection fraction and a seven per cent increase in Vcf in hearts of the same mass as insulin-treated compared to hearts of the same mass as control at a filling pressure of 15 cm H,O. The observed increases in insulin-treated compared to control at 15 cm H,O were six per cent for pressure development, IS:/, for ejection fraction and 17:/, for V,f. Therefore it is unlikely that the enhanced contractile function in insulin-treated relative to control can be totally attributed to a difference in heart mass. Secondly, if insulin-treated were
et al.
hyperinsulinemic, it is possible that insulin in higher concentration may have been available to the insulin receptor during the perfusion in hearts from insulin treated. Although we did not measure plasma insulin levels in insulin-treated animals, plasma glucose levels as low as 50 mg% were observed occasionally which suggests that insulin-treated animals may have been hyperinsulinemic at times. Although others [31] have reported that insulin has no effect on mechanical performance when added to the perfusate, we have observed increases in pressure development with insulin provision [Zl]. When insulin is added to the perfusate, lactate production increases at least two-fold in isolated working hearts [7, 191. These effects of insulin are directionally similar to the differences observed between insulintreated and control (Figure 6). Early studies [S, 211 have reported that insulin washes out of isolated perfused hearts in about 40 min. However, positive differences in contractile function were observed between hearts from insulin-treated and control after any residual insulin would have washed out. Therefore, it is difficult to attribute differences between insulin-treated and control to the presence of a higher insulin concentration in perfused hearts of insulin-treated animals. The studies in Experiment 2 were performed to explore the possibility that hyperinsulinemia may benefit the myocardium. In these studies, cardiac performance did not appear to be enhanced as measured by pump or muscle function indices. However, evidence of a faster relaxation was observed in hearts of hyperinsulinemic animals and this adaptation was reproducible in two separate groups of animals. We have previously demonstrated [N] in hearts of diabetic animals that Ca2+ binding and uptake in sarcoplasmic reticulum vesicles is depressed and this correlates with a slower relaxation in isolated perfused hearts [19] and isolated papillary muscles [IO]. It is possible in these current experiments that sarcoplasmic reticular function was enhanced. Actomyosin ATPase activity was elevated in hearts from the hyperinsulinemic animals similar to the finding in hearts from insulintreated diabetic animals (Table 3). The
Reversal
of Diabetic
mechanism underlying the hyperinsulinemiainduced increase in actomyosin ATPase activity may include alterations in myosin isoenzyme distribution, changes in the regulatory proteins of the thin filament, or phosphorylation of light chain 2 or troponinI. These possibilities deserve further investigation. The discrepancy between the elevated actomyosin ATPase and the absence of a corresponding improvement in pump or muscle function in hearts from hyperinsulinemic animals is unclear. We have previously observed an improvement in cardiac performance in the absence of a change in actomyosin ATPase in hearts of male rats subjected to a chronic running program [18, 281. 0 ne may speculate that no single biochemical mechanism controls cardiac contractility but rather several systems, namely the sarcolemma, the sarcoplasmic reticulum and the contractile proteins, operate in conjunction. In summary, the present studies have demonstrated that the severe diabetic cardiomyopathy induced by injection of streptozotocin is completely reversible by subsequent insulin therapy in vivo. Whether it is the provision of insulin that confers the reversibility or whether a euglycemia provided
Cardiomyopathy
4.57
by other means results in similar findings is not known. The fact that hyperinsulinemia in control animals also seems to affe’ct cardiac actomyosin ATPase suggests that insulin may be specific to the correction. jof the diabetic cardiomyopathic state. The present investigation also confirms the strelptozotocin-induced diabetic cardiomyopathy is a true model of diabetes, since the demonstration of complete reversibility by insulin treatment excludes the possibility of the cardiomyopathy being directly due to strelptozotocin cardiotoxicity. Acknowledgements We wish to thank Gary Ciambrone and Alwyn Murphy for their excellent technical assistance and Mrs Carol Atror for her excellent secretarial assistance. We also wish to thank the Upjohn Co. for their generous donation of streptozotocin. This work was supported by U.S. Public Health Service Grant HL 21482. T. F. Schaible is the recipient of Young Investigator Award HL 25372. This work was presented in part in tlhe Young Investigator Prize Contest, IX World Congress of Cardiology, Moscow, USSR, June 1982.
References 1
2 3 4
5 6 7
8 9 10
BERGMEYER, H. U., BERNT, E., SCHMIDT, F., STORK, H. D-glucose. Determination with hexokiwse and glucose -6-phosphate dehydrogenase. In : Bergmeyer, H. U. (Ed.) Methods of Enzymatic Analysis, pp. I196-1201. New York: Academic Press (1974). BERSOHN, M., SCHEUER, J. Effects of physical training on end-diastolic volume and myocardial performance of isolated rat hearts. Circ Res 40, 510-516 (1977). BERSON, S. A., YALOW, R. S. Insulin in blood and insulin antibodies. Am J Med 40, 676 (1966). BERSON, S. A., YALOW, R. S. General radioimmunoassay. In: Berson, S. A., Yalow, R. S. (Eds) Methods in Investigative and Diagnostic Endocrinology. Part I : General Methodology, pp. 84-120. Amsterdam: North-Holla.nd (1973). BHAN, A. K., SCHEIJER, J. Effects of physical training on cardiac actomyosin adenosine triphosphatase activity. Am J Physiol 223, 1486-1490 (1972). BLEEHEN, N. M., FISHER, R. B. The action of insulin in the isolated rat heart. J Physiol (London) 123, 260-276 (1954). CHAIN, E. B., MANSFORD, K. R. L., OPIE, L. H. Effects of insulin on the pattern of glucose metabolism in the perfused working and Langendorff heart of normal and insulin-deficient rats. Biochem J 115, 537-546 (1969). FACTOR, S. M., BHAN, R., MINASE, T., WOLINSKY, H., SONNENBLICK, E. H. Hypertensive-diabetic cardiiomyopathy in the rat. An experimental model of human disease. Am J Pathol 102, 219-228 (1981). FEIN, F. S., STROBECK, J. E., MALHOTRA, A., SCHEUER, J., SONNENBLICK, E. H. Reversibility of diabetic cardiomyopathy with insulin in rats. Circ Res 49, 1251-1262 (1981). FEIN, F. S., KORNSTEIN, L. B., STROBECK, J. E., CAPASSO, J. M., SONNENBLICK, E. H. Altered myocarclial mechanics in diabetic rats. Circ Res 47, 922-933 (1980).
M.C.C.
458 II 12 13 14 15 16 17 18 19 20 21 22
23 24 25 26 27 28 29 30 31 32 33
34
35
T. F. Schaible
et
al.
Ho~oas~, H. J. L-(.+)-Lactate. Determination with lactic dehydrogenase-and DPN.. In: Bergmeyer, H. U. (Ed.) Methods of Enzymatic Anatysis, pp. 266-270. New York: Academic Press (1963). MALHOTRA, A., PENPARGKUL, S., FEIN, F., SONNENBLICK, E. H., SCHEUER, J. The effect of streptozotocin induced diabetes in rats on cardiac contractile proteins. Circ Res 49, 1243-1250 (1981). MALHOTRA, A., PENPARGKUL, S., SCHAIBLE, T., SCHEUE~, J. Contractile proteins and sarcoplasmic reticulum in physiologic cardiac hypertrophy. Am J Physiol 241, H263-H267 (1981). MILLER, T. B. Cardiac performance of isolated perfused hearts from alloxan diabetic rats. Am J Physiol 236 H808-H812 (1979). MODRAK, J. Collagen metabolism in the myocardium from streptozotocin-diabetic rats. Diabetes 29,547-550 (1980). MURPHY, R. D., VAILAS, A. C., TIPTON, C. M., MATTHES, R. D., EDWARDS, J. G. Influence of streptozotocininduced diabetes and insulin on the functional capacity of rats. J Appl Physiol 50, 482-486 (1981). PAIN, V. M., GARLICK, P. J. Effect of streptozotocin diabetes and insulin treatment on the rate of protein synthesis in tissues of the rat in vim. J Biol Chem 249, 4510-4514 (1974). PENPARGKUL, S., MALHOTRA, A., SCHAIBLE, T., SCHEUER, J. Cardiac contractile proteins and sarcoplasmic reticulum in hearts of rats trained by running. J Appl Physiol 48, 409-413 (1980). PENPARGKUL, S., SCHAIBLE, T., YIPINTSOI, T., SCHEUER, J. The effect of diabetes on performance and metabolism of rat hearts. Circ Res 47, 91 l-921 (1980). PENPARGKUL, S., FEIN, F., SONNENBLICK, E. H., SCHEUER, J. Depressed cardiac sarcoplasmic reticular function from diabetic rats. J Mol Cell Cardiol 13, 303-309 (1981). RANNELS, D. E., HJALMARSON, A. C., MORGAN, G. E. Effects of non-carbohydrate substrates on protein synthesis in muscle. Am J Physiol 226, 538-539 (1974). REGAN, T. J., ETTINGER, P. O., KHAN, M. I., JESRAN, M. U., LYONS, M. M., OLDEWURTEL, H. A., WEBER, M. Altered myocardial function and metabolism in chronic diabetes mellitus without ischemia in dogs. Circ Res 35, 222-237 (1974). REGAN, T. J., LYONS, M. M., AHMED, S. S., LEVINSON, G. E., OLDEWURTEL, H. A., AHMED, M. R., HAIDER, B. Evidnece of cardiomyopathy in familial diabetes mellitus. J Clin Invest 60,885-899 (1978). REGAN, T. J., WV, C. F., YEH, C. K., OLDEWURTEL, H. A., HAIDER, B. Myocardial composition and function in diabetes. The effects of chronic insulin use. Circ Res 49, 1268-1277 (1981). SANDERSON, J. E., BROWN, D. J., RIVELLESE, A., JOHNER, E. Diabetic cardiomyopathy? An echocardiographic study of young diabetes. Br Med J 1, 404-407 (1978). SCHAIBLE, T. F., SCHEIJER, J. Effects of physical training by running or swimming on ventricular performance of rat hearts. J Appl Physiol46, 854-860 (1979). SCHAIBLE, T. F., SCHEUER, J. Cardiac function in hypertrophied hearts from chronically exercised female rats. J Appl Physiol 50, 1140-l 145 (1981). SCHAIBLE, T. F., PENPARGKUL, S., SCHEUER, J. Differences in male and female rats in cardiac conditioning. J App Physiol 50, 112-117 (1981). SCHAIBLE, T. F., MALHOTRA, A., SCHEUER, J. The effect of rat heart size on cardiac performance (Abstract). Fed Proc 41, 1013 (1981). SEGAL, S. A., BLAIR, A. E., WYNGAARDEN, J. B. An enzymatic spectrophotometric method for the determination of pyruvic acid in blood. J Lab Clin Med 48, 137-143 (1956). TAEGTMEYER, H., HEMS, R., KREBS, H. A. Utilization of energy-providing substrates in the isolated working rat heart. Biochem J 186, 701-711 (1980). UMBREIT, W. W., BURRIS, R. H., STAUFFER, J. F. Manornetric techniques (3rd edn) Minneapolis, MN: Burgess, p. 5 (1957). YALOW, R. S., BERSON, S. A. Insulin: radioimmunoassay. In: Berson, S. A., Yalow, R. S. (Eds) Methodsin Part III: Non-pituitary Hormones. pp. 834-870. Amsterdam: Investigative and Diagnostic Endocrinology. North-Holland (1973). YIN, F. C. P., SPURDEON, H. A., WEISFIELDT, M. L., LAKATTA, E. G. Mechanical properties of myocardium from hypertrophied rat hearts. A comparison between hypertrophy induced by senescence and by aortic banding. Circ Res 46, 292-300 (1980). ZAR, J. H. Biostatistical Analysis. Englewood Cliffs, N. J. : Prentice-Hall (1974).
Cell
Left
Thomas
Cardiol
15, 445-458
Ventricular
(1983)
Function in Diabetic
F. Schaible,
Ashwani
After Chronic and Normal
Malhotra,
William
Insulin Rats
Treatment
A. Bauman
and James
Scheuer
Departments of Medicine and Physiology and Biophysics. Albert Einstein College of Medicine, and The Division of Cardiology and Department of Clinical Sciences, MonteJiore Hospital and Medical Center, Bronx, New York 10467, USA. (Received 3 August 1982, accepted in revised form 17 January
1983)
T. F. SCHAIBLE, A. MALHOTRA, W. A. BAUMAN AND J. SCHEUER. Left Ventricular Function After Chronic Insulin Treatment in Diabetic and Normal Rats. Journal of Molecular and Cellular Cardiology (1983) 15, 445458. Previous reports have documented a cardiomyopathy in rats resulting from streptozotocininduced diabetes. In order to determine the reversibility of streptozotocin-induced cardiomyopathy to insulin therapy, hearts from rats made diabetic by streptozotocin for 6 weeks and then treated with insulin for 3 weeks were compared with untreated diabetic rats and control rats not injected with streptozotocin. When perfused in an isolated working heart apparatus with 5.5 mM glucose as substrate, hearts from untreated diabetic rats when compared to hearts from either streptozotocin-injected rats treated with insulin or control rats showed significant depressions in peak left ventricular pressure, maximal positive and negative dP/dt, oxygen extraction, lactate production and effluent lactate; pyruvate ratio. Ca2+-actomyosin ATPase was also depressed in untreated diabetics. As left atria1 pressure was raised in untreated diabetic rats, a decline in cardiac output was observed, whereas in insulin-treated or control groups there was no such negative response. Indices of cardiac performance were significantly greater in insulin-treated rats when compared to control rats suggesting overcorrection with insulin therapy. To explore whether insulin treatment may have a beneficial effect on the myocardium control rats were made hyperinsulinemic for 6 to 7 weeks. Shorter isovolumic relaxation times and elevated values for Ca2+actomyosin ATPase were observed in hearts from hyperinsulinemic animals when compared to hearts from control animals. These results demonstrate complete reversibility of streptozotocin-induced cardiomyopathy and confirm that this condition is due to insulin deficiency and not to a primary cardiotoxic effect of streptozotocin. KEY WORDS: insulinemia.
Diabetes
mellitus;
Ventricular
function;
Introduction Insulin deficient diabetes has been associated with a specific cardiomyopathy in humans [23, 251 and experimental animals [IO, 14, 19, 221. Most animal studies have relied on chemical substances as diabetogenic agents which purportedly selectively destroy the beta cells of the pancreas, the primary source of insulin synthesis. Previous studies have seldom incorporated a diabetic group treated with insulin in the experimental design so that untoward effects of the agent may be assessed. In some studies that have included such a group, only partial reversal of diabetesrelated abnormalities has occurred with insulin treatment [IS, 17, 241. OOZZ-2828/83/070445+ M.C.C.
14 $03.00/O
Actomyosin
ATPase;
Insulin
therapy;
Hyper-
A previous report from this laboratory [19] characterized a cardiomyopathy in rats made diabetic for eight weeks by a single injection of streptozotocin. Isolated working hearts from streptozotocin-injected animals showed depressed cardiac contractile function and impaired relaxation, despite normal coronary flows and myocardial oxygen consumptions. In order to determine the extent to which the cardiac abnormalities found in streptozotocin diabetes could be reversed, the present study was designed using a streptozotocin injected group that was subsequently treated daily with insulin. Our results demonstrated that the cardiomyopathy associated with streptozotocin-induced diabetes could 0
1983 Academic
Press Inc.
(London)
Limited u
446
T. F. Schaible
be reversed by insulin therapy. In addition, hearts from streptozotocin diabetic rats that were treated with insulin demonstrated better performance than hearts from control animals that were not injected with streptozotocin. Therefore, a second study was designed to determine whether chronic insulin treatment would have a beneficial effect on the myocardium.
Methods
Experimental
groups
Experiment 1: The e$ects of insulin treatment on streptozotoci’n-induced diabetic cardiomyopathy. Male Wistar rats initially weighing 180 to 200 g were fasted overnight and then were made diabetic the following morning by injecting streptozotocin, 50 mg/kg, dissolved in 0.05 M citrate, pH 4.5, into the tail vein. Control rats from the same initial group were not injected. All animals were fed Purina Rat Chow and watered ad libitum for the duration of the study. Six weeks after injection the diabetic group was subdivided into a group that received insulin treatment and a group that did not receive treatment. Animals were placed in the insulin-treated and non-treated diabetic groups according to similar blood glucose levels and body weights. Protamine zinc insulin (Lilly) was injected subcutaneously at approximately 4 p.m. every day. Blood glucose and body weight were measured in all of the insulin-treated animals at 3 to 4 day intervals and in some of the non-treated and control animals at weekly intervals. Blood samples were drawn at 9 to 10 a.m. from the tail vein of conscious restrained rats. Rats in the insulin-treated group received treatment (3 to 6 U/day) for 18 to 30 days at which time they were sacrificed and their hearts were studied along with hearts from rats in the non-treated diabetic and control groups. Rats in the insulin-treated group did not receive insulin for 18 h prior to the time their hearts were studied. Experiment 2: The e$ect of hyperinsulinemia on cardiac pe$rmance. Hyperinsulinemia was produced in two groups of rats. The first group of rats (CI,) weighed approximately 400 g when daily insulin injections were
et al.
started. The second group of rats (CI,) weighed approximately 200 g when treatment began. Rats in both groups were injected initially with 4 U Protamine zinc insulin daily. This dosage was progressively increased to 7 U daily over the course of the study. Both hyperinsulinemic groups received a 7.5:/, (w/v) dextrose solution as drinking water to avoid deaths from hypoglycemia. Treatment lasted for 40 to 50 days at which time the hearts from treated rats were studied and compared to hearts from age-matched untreated rats (C, and C,) . During the course of the study blood samples were drawn from the tail vein to determine glucose and insulin levels. Details for the timing of insulin injection and blood collection were the same as in Experiment 1. Heart perfusions: Full descriptions of the isolated working rat heart apparatus have been published previously [2, 26, 271. The perfusate was a modified Krebs-Henseleit buffer at 37°C gassed with a 95:/, 0, and 5!/, CO, mixture and containing 5.5 mM glucose, and 2.0 mM calcium with 0.5 mM EDTA yielding 1.5 mM free calcium. The perfusate was not recirculated. Hearts in Experiment 1 were perfused without insulin while hearts in Experiment 2 were perfused with a buffer containing 0.01 U/ml regular insulin (Lilly). Insulin was included in the buffer in Experiment 2 to avoid the possibility that a higher residual insulin concentration in hearts from hyperinsulinemic animals would contribute to improved function. Left ventricular pressure was measured through a 2.5-cm polyethylene (PE-60) catheter inserted through the apex of the heart and attached to a Statham P23dB strain gauge pressure transducer. A second catheter was placed through the left ventricular apex for dye injection. Aortic pressure was measured from a sidearm on the aortic cannula approximately 7 to 8 mm above the aortic valve. The frequency responses of the pressure measuring systems were flat f lo:/, to 30 Hz. Instantaneous aortic ilow was measured from a cannulating 2.5 mm I.D. flow probe (Statham-Gould) inserted in the aortic outflow tubing. Coronary flow was measured directly as right heart outflow, and cardiac output was measured as aortic flow plus coronary flow. Dye concentrations in the aorta were
Reversal
of Diabetic
measured by a densitometer system placed into the aortic cannula. All hearts were paced from the right atrium at a rate of 340 beats/ min. Oxygen tension was measured in the perfusate from the left atria1 reservoir and from a pulmonary arterial catheter. Arteriovenous differences in oxygen tension were converted to oxygen consumption by multiplying the arteriovenous oxygen tension by coronary flow and the appropriate Bunsen coefficient 1321 and dividing by left ventricular dry weight. Samples were taken from the atria1 reservoir and right ventricular outflow for analysis of lactate and pyruvate. Whenever possible, one heart from each group within an experiment was perfused on a given day. Cardiac function was assessed as a function of preload, which was varied by changing the height of the reservoir filling the left atrium (left atria1 pressure). The height of the aortic column was 80 cm at all times. After a 10 to 15 min period of retrograde during which catheters for left perfusion, ventricular pressure and dye injection were positioned, antegrade perfusion was begun by unclamping the left atria1 cannula and the heart was allowed to equilibrate for 15 min at a left atria1 pressure of 10 cm H,O. After an initial recording of data at 10 cm HZ0 the filling pressure was lowered to 5 cm H,O, then raised in sequence to 10, 15, and 20 cm H,O and finally lowered to 10 cm H,O, all for periods of 7.5 min. After 5 min at each filling pressure, records were made of dynamics, multiple dye dilution curves were recorded and the coronary flow and cardiac output were measured over a I-min period. All analog data (dye concentration, aortic flow and left ventricular and aortic pressure) were stored on magnetic tape for later analysis on a digital computer. End-diastolic volumes were estimated from the dye-dilution curves. A full description of this method and the validation of its accuracy for measuring ventricular volumes have been published previously [2]. End-diastolic volume was calculated by -dividing the directly measured stroke volume (cardiac output divided by heart rate) by the ejection fraction determined from the multiple dye curves. Flow and pressure analog data were M.C.C.
44.7
Cardiomyopathy
digitized at 660 samples/s and the measurements of cardiac dynamics were determined as previously described [2, 271. Geometric calculations were based on the assumption that the left ventricule has a spherical geometry with a constant wall thickness as described presriously [27]. By integrating aortic flow during ejection, the ejected volume could be subtracted from the enddiastolic volume, yielding instantaneous left ventricular volume. This permitted calculation of the velocity of circumferential fiber shortening ( V,$, total circumferential fiber shortening, and fractional shortening at th.e midwall. Instantaneous wall stress was estimated from the La Place equation [27] and The isovolumic reexpressed in g/cm. laxation time was calculated as the period from the onset of the dicrotic notch (aortic valve closure) until the left ventricular pressure relaxed to the left atria1 pressure (mitral valve opening). At the end of each experiment the atria and great vessels were dissected free and the right ventricular free wall was removed. The left ventricle (including the septum) wals weighed to determine wet weight. A small piece of the left ventricle (approximately 0.2 g wet weight) was dried to constant weight in an oven to calculate left ventricu1a.r dry weight. The remainder of the left ventricule was retained for analysis of actomyosin ATPase. Actomyosin A TPase All hearts were stored at -80°C in 5OqJ, glycerol containing 50 mM KC1 and 10 rnlti KPO,(pH 7.0) prior to preparing extracts. Hearts of experimental animals were always extracted and analyzed simultaneously with hearts of controls using the same reagents and incubation conditions. Actomyosin was used because this permits the evaluation of muscle from individual hearts, rather than necessitating the pooling of several hearts which is required when pure myosin is studied. The measurement of actomyosin ATPase activity in high ionic strength (0.3 M KCl) is similar to measuring pure myosin, and in severaL studies from our laboratory there has been excellent correspondence without exception, between this measurement, calcium activated
448
T. F. Schaible
ATPase and actin activated myosin ATPase [12, 133. The methods of preparing and analyzing cardiac actomyosin from individual hearts have been detailed previously [5]. Results are expressed as pmol Pi liberated per min per mg protein at 30°C. Chemical analyses Lactate and pyruvate were determined in perfusate samples by the methods of Hohorst [11] and Segal [30] respectively. Glucose in 37; perchloric acid extracts from blood was determined by a hexokinase : glucose-6phosphate dehydrogenase method [I]. The concentration of insulin in the plasma samples was determined by radioimmunoassay using two guinea-pig antisera with different sensitivities and species-specificities, according to methods previously described [3, 4, 331. The assay system employing GP 11-8-14 had a minimal sensitivity for the detection of beef insulin of 10 pg/ml incubation mixture. Rat insulin crossreacted tenfold more weakly in this system. Plasma insulin in the beef-insulin treated animals was assayed with this antiserum employing 12sI-beef insulin as the tracer and Lilly beef insulin as standard. Since endogenous rat insulin would be suppressed by insulin treatment, none would be measured in this system. The assay system employing 1251-pork insulin as the tracer and GP N-3-13 was capable of detecting 3 pg Lilly rat insulin/ml and was used for determination of endogenous rat insulin in non-insulin-treated animals. Each plasma sample was assayed at a dilution of plasma 1: 20 or greater. There were no detectable anti-insulin antibodies at these plasma dilutions. Tibia1 lengths When the animals were sacrificed, both tibia bones were dissected out. These were dried in an oven and then measured to the nearest 0.1 mm under a magnifying glass. The length was taken as the distance from the condyles to the tip of the medial malleolus [34]. The dry left ventricular weight to dry tibia1 length ratio was compared to the dry left ventricular weight to body weight ratio for each group of animals in Experiment 1.
et al.
Statistical
anaCysis
Results at any given filling pressure for the three groups of animals in Experiment 1 were first submitted to a one way analysis of variance [35] to test for any group differences. If a significant F value was obtained (P < 0.05), differences between any two groups were tested by the Neuman-Keuls multiple comparison test [35]. In Experiment 2 where only two groups were compared the unpaired student t test was employed [35]. Significant differences at the P < 0.05 and P < 0.001 level are reported.
Results
Experiment
1
Figure 1 shows the time course changes in blood glucose (upper panel) and body weight (lower panel) for the control, untreated diabetic rats and streptozotocin-injected rats treated with insulin groups. Insulin dosages for the treatment schedule are also shown. After 5 days using 5 to 6 U/day protamine zinc insulin, glucose values in the insulintreated group declined to values in the control group and remained at this level using dosages of 3 to 4 U/day. Values for the untreated diabetic group remained above 400 mg%. Body weights in the insulintreated group increased substantially once insulin therapy was initiated. Table 1 shows some of the gross morphological measurements in the three groups. For measurements of dry heart weight, body weight and dry tibia1 length, the three groups were statistically autonomous and values for insulin-treated were intermediate between values for control and untreated diabetic groups. For the dry heart weight to body weight ratio, values were similar between untreated diabetic and insulin treated groups and both of these were greater than the value for control. Conversely, for the dry heart weight to tibia1 length ratio, values were similar between control and insulin-treated and both of these were greater than the value for untreated diabetic group. Therefore, heart size relative to body size was reversed in insulin treated diabetes only when expressed as the dry heart weight to tibia1 length ratio.
Reversal
of Diabetic
Cardiomyopathy
44:9
&/450
-
400
i/-
-
s i .P $ p” 4
FIGURE 1. Values for blood glucose and body weight in diabetic, diabetic treated with insulin and control animals during the course of insulin treatment. The diabetic and treated diabetic groups had been diabetic 6 weeks prior to the beginning of insulin treatment. Daily insulin dosages during the treatment schedule are shown at the bottom. Results for body weight for all groups and for glucose in the treated diabetic group represent the mean i SAM. for 10 to 17 animals. Glucose values for the diabetic group and control group, represent the average of values for 3 to 4 animals. O----a, control; A’- - -[l, diabetic; A- - -A, treated diabetic.
350
-
300
-
250
-
i,-“*
O-
_A
.,i-----i’ I- -
,,I-
**i.---
,’ _ -,/_____-- -----&+
_________ A ___. - -----
I
I
0
4
I 8
I I2
/-- i
_/ -
a
-----------
I 16
I 20
24
Days k-6-4-5
a-3-4 Insulin
TABLE
1. Results
for
body
weight,
dose
!U:
heart
weight
and
tibia1
length
DHW/BW n Experiment
Cd
DHW
(m.d
DHW/DTL DTL
bgld
11 11 11
Experiment
491 f 12” 296 f 7 375 & ll”,d
233 & 5b 17064 210 -& 6b+
0.475 0.575 0.562
f f f
0.009b 0.010 0.008d
4.18 3.76 3.89
f f f
0.030 0.030 0.027-
55.8 44.8 54.0
f l.lb & 0.9 & 1.3b
2
G
7
498
f
20
241 &
15
0.482
f
0.018
7
495
f
11
242 f
4
0.490
*
0.014
4
CI,
(mdcm)
1
CI, Group G
(cm)
1
c D DI Group
BW
-
-
2 7
390
-& 9
184 f
7
404
f
201 & 3”
5
0.472
& 0.007
-
0.495
f
-
0.007”
Values arc mean & S.E. BW, body weight; DHW, dry heart weight; DTL, dry tibia1 length. For Experiment 1: &P < 0.05, bP < 0.001, for C v. D or DI v. D; c,P < 0.05, *P < 0.001 for DI v. C. For Experiment 2: a P < 0.05 for C, v. CI,. C, control; D, untreated diabetic; CI, insulin-treated diabetic.
-
450
T. F. Schaible
Measurements of cardiac output and stroke work (both per g dry left ventricle) are shown in the top panels of Figure 2. Hearts from the insulin-treated group demonstrated a significantly higher cardiac output than both untreated diabetic and control groups at the highest left atria1 pressure. Stroke work for the insulin-treated group was significantly greater than control and untreated diabetic groups at 15 and 20 cm atria1 pressure, but, in addition, values for control were greater than values for the untreated diabetic group at the highest left atria1 pressure. As atria1 pressure was increased from 15 to 20 cm H,O values for both cardiac output and stroke work decreased in the untreated dia-
et al.
betic group (P < O-005), remained the same in controls (P > 0.05) and increased in the insulin-treated group (P < 0.005). Values for peak left ventricular systolic pressure and maximum positive dP/dt are shown in the bottom panels of Figure 2. Values for both of these functions were significantly greater in the control and insulin-treated group compared to untreated diabetics at all left atria1 pressures. At some atria1 pressures, peak left ventricular systolic pressure was greater in the insulin-treated group than control. Figure 3 shows data for two measurements of ventricular relaxation, namely the maximum rate of left ventricular pressure decline (max - dP/dt) and the isovolumic relaxa-
1.25
-
1.00
-
$j
f
Left
atrial
filling
pressure
(cm
H:! 0)
FIGURE 2. Measurements of pump performance as a function of left atria1 filling pressure for the groups in experiment 1. Cardiac output and stroke work are shown in the top panels and are normalized for dry left ventricular weight. Results represent the mean & 1 S.E. In addition, paired t statistics showed a significant increase (P < 0.05) in cardiac output and stroke work going from 15 to 20 cm Hz0 atria1 pressure for hearts from treated diabetic animals, no change (P > 0.05) in hearts from control animals, and a significant decrease (P < 0.05) in hearts from diabetic animals. Pressure related measurements from hearts in Experiment 1 are shown in the bottom panels. PLVSP = peak left ventricular systolic pressure; Max + dP/dt = maximum rate of left ventricular pressure rise. e--e, control (11) ; ,& - -A, diabetic (11) ; ); - -A, treated diabetic (11). < 0.001 for control v. untreated Numbers in parentheses indicate the number of preparations. *P < 0.05, . **P diabetic or insulin-treated diabetic v. untreated diabetic; +P < 0.05 for insulin-treated diabetic v. control.
Reversal
of Diabetic
I IO Left
atrial
FIGURE 3. Measurements of left ventricular maximum rate of left ventricular pressure decline; the same as in Figure 2.
filling
pressure
1”
3 L$
ij
E ; P :: -I5
2.5 2.0
4 01‘ IO’
I 5
I IO
I I5
I 20
atria1
I 20
I lo-
(cm l-l, 0)
All
1. Max - dP/dt == other symbols are
200 175 150
a 4
125
O-
I IO Left
I 15
V,f
::
I -I
I IO
similar in untreated and diabetic control groups but values at high left atria1 pressure were significantly greater in insulin-treated compared to either untreated diabetic or control groups. Fractional shortening calculated at the midwall showed patterns of differences that were similar to those observed for ejection fraction. Maximum was greater in insulin-treated than both control and untreated diabetic groups at all left
“6 3.0
I 5
relaxation in hearts from Experiment Isov. relax = isovolumic relaxation.
tion period. Values for control and insulintreated groups were similar and significantly different from values for the untreated diabetic group. The isovolumic relaxation period was nearly twice as long in the untreated diabetic group as compared to that observed in insulin-treated or control groups. Figure 4 shows values for ejection fraction and derived measurements for shortening, force and velocity. Ejection fraction was
.-:
451
Cardiomyopathy
filling
FIGURE 4. Measurements offorce, velocity and shortening velocity of circumferential fiber shortening. All other symbols
pressure
(cm Hp 0)
in hearts from Experiment are the same as in Figure
1. Max 2.
V,f = maximum
452
T. F. Schaible
atria1 pressures. However, maximum wall stress was similar in insulin-treated and control and greater than in the untreated diabetic group at the higher filling pressures. Figure 5 shows that end-diastolic pressure was Iower in the untreated diabetic group than either control or insulin-treated groups at all but the lowest left atria1 pressure. Similar relationships amongst the three
et al.
groups were observed for end-diastolic wall stress, although these data are not shown. Figure 5 also shows that end-diastolic volume (per g dry left ventricle) was not different amongst the three groups. Data describing oxygen delivery and utilization are shown in Table 2. Hearts from the untreated diabetic group demonstrated greater coronary flows per g at all left atria1
6-
3-
o-
I
I
I
I
I
I
IO
5
IO
15
20
IO Left
FIGURE 5. End-diastolic in hearts from Experiment as in Figure 2.
TABLE
2. Values for Experiment
coronary 1
oxygen
flow,
Left
Oxygen
C D DI Myocardial C D DI
Jaw
(ml/g/min) 87 f 3” 98 zt 4 84 f 2”
extraction 0.642 0.561 0.669
filling
pressure
extraction
atria1 pressure 10
92 f 3” 104 + 3 90 f 2a
I 5
’
IO (cm
pressure (EDP) and end-diastolic volume 1. EDV is expressed per g dry left ventricular
5 Coronary C D DI
atria1
01
I IO
I 15
I 20
L I(
Hz 0)
as functions (EDV) weight. All other
and
oxygen
of left atria1 pressure symbols are the same
consumption
for
hearts
in
(cm H,O) 15
20
92 5 38 104 f 4 90 f 2”
92 + 3” 106 f 3 91 * 3a
(ml/ml) + 0.014s + 0.017 * 0.011”
oxygen consumption 1.10 f 0.03 1.09 & 0.02 1.12 * 0.02
0.667 0.593 0.717
f 0.012b -& 0.014 + 0.Ollb.c
(mllglmin) 1.21 & 0.04 1.22 * 0.03 1.29 * 0.03
0.684 0.598 0.748
& 0.013b 5 0.015 & 0.012b.c
1.24 f 1.24 j, 1.35 *
0.03 0.03 0.04
Values represent the mean & 1 S.E. of 11 hearts. Coronary flow and oxygen g dry weight. BP < 0.05; bP < 0.001 C v. D or DI v. D.; CP < 0.05 DI v. C. Abbreviations: see Table 1.
0.697 0.591 0.748
& 0.012” & 0.017 & 0.017bsc
1.25 f 1.24 + 1.36 * consumption
0.03 0.03 0.04
are expressed
per
Reversal
of Diabetic
pressures when compared to either insulintreated or control groups. Oxygen extraction was less in the untreated diabetic group compared to either insulin-treated or control groups and it was less in control than in insulin-treated groups. Myocardial oxygen consumption was similar in all three groups but there was a trend towards higher values in insulin treated compared to either control or untreated diabetic groups. Values for lactate and pyruvate production are shown in Figure 6. Hearts from the untreated diabetic group produced larger amounts of pyruvate and lesser amounts of lactate when compared to either control or insulin-treated groups. In addition, lactate production and the sum of lactate and pyruvate production were greater in insulintreated than control groups at the higher left atria1 pressures. The ratio of lactate to pyruvate production was substantially less in
Cardiomyopathy
the untreated diabetic group when compared to either control or insulin-treated groups. Table 3 shows values for Ca2+ stimulated actomyosin ATPase for the three groups in Experiment 1. ATPase activity was approximately 50°j, depressed for the untreated diabetic group compared to either control or insulin-treated groups. There was a significant 17:/:, elevation in enzyme activity in the insulin-treated group when compared to control.
Experiment2 Blood glucose and plasma insulin levels obtained during the second and fourth weeks of insulin treatment are shown for treated and untreated animals from Group 2 in Table 4. Plasma insulin levels were four to six times greater in insulin-treated animals at both time points. Blood glucose levels were not different between C, and CI, at the
1
T+
8-
4513
I
I
I
I
I
** l ---____ I **
12 (u
6-
I ** &./I** z /-I 0 * * L6-r* ?----*5
1
5
IO
15
20
IO Left
All A-
30
0, IO
I** I**
Y5 5 9-
-I
atria1
filling
I
*n
pressure
4
~ ______
a _--- ---A-------4
I IO
I 5
I IO
I 15
4 1 20
I IO
(cm H,O)
FIGURE 6. Values for lactate and pyruvate production, their sum and their ratio for hearts in Experiment values are expressed per g dry left ventricular weight. O---e, control (9); A- --A, diabetic - -A, treated diabetic (10).
1. (9);
454 TABLE
T. F. Schaible
3. Gas+-Actomyosin
ATPase
et al.
values for groups in Experiments
n
1 and 2
Ca2+-Actomyosin ATPase (pm01 Pi/mg/min)
Exfieriment 1 C D DI
7 9 6
E@eriment 2 Group 1 Cl CI,
7 10
0.576 & 0.017 0.678 & 0.013”
Groufi 2 G CIZ
8 8
0.539 & 0.015 0.690 i 0.04Za
0.566 & 0.017b 0.281 5 0.017 0.680 & 0.028bsc
Values are mean & S.E.; n = number of hearts. For Experiment 1: b&’ < 0.001 for C v. D or DI v. D; CP < 0.05 for DI v. C. For Experiment 2: BP < 0.005; bp < 0.001 for C1 v. CI,, or C, v. CI,. Abbreviations: see Table 1.
TABLE
4. Blood glucose and plasma insulin levels from control and insulin treated animals in Group 2 from Experiment 2 4 weeks
2 weeks G C* c12
106 h 4 (5) 82 i 11 (‘5)
I
G
19 & 4 (5) 87 & 9” (‘1)
126 f 7 (5) 58 f 6” (13)
I 141 1 (4) 97 * 14” (13)
Samples were obtained during the second and fourth week of insulin treatment. Values are means f 1 S.E. Number of observations are shown in parentheses. G = blood glucose (mg/lOO ml blood); I = plasma insulin (pU/ml plasma). =P < 0.005; bP < 0.001 CI, v. Cz. Abbreviations : see Table 1.
two-weeksamplingpoint butwere significantly lower in CI, at the 4 week sampling point. Table 1 shows data for heart and body weights from insulin-treated and control animals. Heart and body weights were similar between C, and CI, in Group 1. However, in Group 2 where the treatment was started at an earlier age, animals from the treated group (CI,) had mildly elevated heart weights and heart weight to body weight ratio when compared to the untreated group (Cs) .
Data for performance of isolated hearts are shown in Table 5. All flow, volume and contractile measurements were similar in comparing treated and untreated groups in either Group 1 or Group 2. The only consistent differences observed involved measurements of cardiac relaxation. Thus, in Group 1, CI, demonstrated a significantly greater rate in the decline of left ventricular pressure (max - dP/dt) when compared to C, while for both Group I and 2, insulin-treated
Reversal TABLE
5. Dynamic
performance
of Diabetic
in isolated
hearts Group
455
Cardiomyopathy from
Experiment
2 Group
1
2
LAP (cm H@) Coronary (mlidmin)
flow
15 20
Cardiac (ml/g/min)
Output
15 20
87 f 87 f
2 2
517 & 25 525 & 2.5
673 f 676 *
23 24
639 5 22 647 & 25
8.1 * 0.4 9.6 & 0.9
8.7 f 10.5 i
0.6 0.8
8.2 & 0.4 9.0 & 0.5
8.3 + 0.3 8.7 f 0.1
Peak LV Pressure (mmHg)
15 20
104 f 103 *
107 f 107 f
2 2
108+ I07f
113 & 3 112 f 3
Ejection
15 20
0.51 0.52
f 0.03 3 0.04
0.58 0.57
f 0.02 & 0.01
0.63 0.62
3 0.04 f 0.04
0.60 0.60
Max-dP/dt
15
(mm%/s)
20
2720 2610
f 103 + 92
3060 2910
f 136 & 103a
2790 2680
* 128 & 132
2530 2420
Iso”
15 20
17.2 & 1.2 17.6 & 1.7
& 0.03 * 0.08
12.7 f 1.0a 12.6 & l.la
3.18 3.23
rt: 0.16 f 0.14
1 I
19.5 + 0.9 20.3 & 1.5
3.22 3.30
f i
4 3
15 20
2 2
2.69 2.82
103 f 105 f
End-diastolic Pressure (mmHg)
Relax
0.18 0.25
22 24
3 3
15 20
Time (ms)
f *
530 f 551 *
107 f 106f
End-diastolic Volume (ml/g)
fraction
3.02 3.04
83 & 3 86 f 3
0.23 0.32
i 0.03 & 0.04 * *
72 52
14.2 & O.Yb 14.4 * 0.7b -
Values represent the mean & 1 SE. of 7 hearts in each group. LV. LAP = left atria1 filling pressure. “I’ < 0.05; bP < 0.005 for C1 v. CI, or C, v. CI,. Abreviations: see Table 1.
groups had significantly shorter insvolumic relaxation times when compared to their respective untreated group. Table 3 summarizes values for Ca2+stimulated actomyosin ATPase for the groups in Experiment 2. Significantly higher values for enzyme activity in treated animals were observed for the comparisons C, v. CI, and C, v. CI,. These differences amounted to 18% and 28:/, respectively.
Discussion
In the present study, treatment of the streptozotocin diabetic animals with insulin completely reversed all mechanical and metabolic abnormalities observed in isolated working hearts from streptozotocin-diabetic animals. These findings in addition to the finding that depressed levels of cardiac actomyosin ATPase were also reversed in insulin therapy, provide evidence that the streptozotocin effect was not one of direct
Flow
and volume
data
are expressed
per g dlry
toxicity on the heart but related to a reversible diabetic state. The present findin.gs are directionally similar to those reported by Fein et al. [9] who found partial reversal of mechanical abnormalities in papillary muscles after 10 days of insulin treatment in female diabetic rats and complete reversal after 28 days of insulin treatment. This wa.s accompanied by normalization of contractile protein enzyme activity in the same time frame. In a recent study by Murphy et al., [1S] male rats made diabetic with streptozotocin and subsequently treated with 2 U protamine zinc insulin daily for 4 weeks, did not fully recover maximal heart rate response or performance time in a graded treadmill exercise test. The discrepancy between these findings and our own may be due to the possibility that factors that control heart rate may not be completely reversed by insulin therapy in streptozotocin-diabetic rats. The myocardial interstitial fibrosis that occurs in alloxan diabetic dogs is not affected
456
T. F. Schaible
by insulin treatment [24], but there is no fibrosis in streptozotocin diabetic rats [8, 1.51. Thus, diabetic cardiomyopathies involving interstitial fibrotic abnormalities may not be as amenable to reversal by insulin therapy as the diabetic cardiomyopathy in this study which primarily involves contractile dysfunction. Particularly notable in the present studies is evidence that treatment of diabetic animals with insulin resulted in cardiac performance that was greater than observed in control hearts. Values for cardiac output, stroke work, peak pressure development, ejection fraction, fractional shortening and maximal were significantly greater in insulinVCf treated than in control groups. In addition, values for cardiac output and stroke work significantly increased in insulin-treated when the left atria1 pressure was raised from 15 to 20 cm H,O while values in control remained the same. These findings may represents true improvements in contractile performance end-diastolic since end-diastolic pressure, volume and maximal wall stress were similar in insulin-treated and control groups. However, two factors related to our methodology may contribute, in part, to these differences. The first of these involves the differences in heart mass between insulin-treated and control groups, which was 10% less in insulin-treated than control. We have previously shown that small hearts demonstrate higher values for cardiac output per g when compared to large hearts from older animals [19]. However, force, velocity and shortening measures of contractile performance are not as dependent on heart mass as normalized cardiac output. Our studies [29) would predict no differences in peak pressure development, a 4:(, increase in ejection fraction and a seven per cent increase in Vcf in hearts of the same mass as insulin-treated compared to hearts of the same mass as control at a filling pressure of 15 cm H,O. The observed increases in insulin-treated compared to control at 15 cm H,O were six per cent for pressure development, IS:/, for ejection fraction and 17:/, for V,f. Therefore it is unlikely that the enhanced contractile function in insulin-treated relative to control can be totally attributed to a difference in heart mass. Secondly, if insulin-treated were
et al.
hyperinsulinemic, it is possible that insulin in higher concentration may have been available to the insulin receptor during the perfusion in hearts from insulin treated. Although we did not measure plasma insulin levels in insulin-treated animals, plasma glucose levels as low as 50 mg% were observed occasionally which suggests that insulin-treated animals may have been hyperinsulinemic at times. Although others [31] have reported that insulin has no effect on mechanical performance when added to the perfusate, we have observed increases in pressure development with insulin provision [Zl]. When insulin is added to the perfusate, lactate production increases at least two-fold in isolated working hearts [7, 191. These effects of insulin are directionally similar to the differences observed between insulintreated and control (Figure 6). Early studies [S, 211 have reported that insulin washes out of isolated perfused hearts in about 40 min. However, positive differences in contractile function were observed between hearts from insulin-treated and control after any residual insulin would have washed out. Therefore, it is difficult to attribute differences between insulin-treated and control to the presence of a higher insulin concentration in perfused hearts of insulin-treated animals. The studies in Experiment 2 were performed to explore the possibility that hyperinsulinemia may benefit the myocardium. In these studies, cardiac performance did not appear to be enhanced as measured by pump or muscle function indices. However, evidence of a faster relaxation was observed in hearts of hyperinsulinemic animals and this adaptation was reproducible in two separate groups of animals. We have previously demonstrated [N] in hearts of diabetic animals that Ca2+ binding and uptake in sarcoplasmic reticulum vesicles is depressed and this correlates with a slower relaxation in isolated perfused hearts [19] and isolated papillary muscles [IO]. It is possible in these current experiments that sarcoplasmic reticular function was enhanced. Actomyosin ATPase activity was elevated in hearts from the hyperinsulinemic animals similar to the finding in hearts from insulintreated diabetic animals (Table 3). The
Reversal
of Diabetic
mechanism underlying the hyperinsulinemiainduced increase in actomyosin ATPase activity may include alterations in myosin isoenzyme distribution, changes in the regulatory proteins of the thin filament, or phosphorylation of light chain 2 or troponinI. These possibilities deserve further investigation. The discrepancy between the elevated actomyosin ATPase and the absence of a corresponding improvement in pump or muscle function in hearts from hyperinsulinemic animals is unclear. We have previously observed an improvement in cardiac performance in the absence of a change in actomyosin ATPase in hearts of male rats subjected to a chronic running program [18, 281. 0 ne may speculate that no single biochemical mechanism controls cardiac contractility but rather several systems, namely the sarcolemma, the sarcoplasmic reticulum and the contractile proteins, operate in conjunction. In summary, the present studies have demonstrated that the severe diabetic cardiomyopathy induced by injection of streptozotocin is completely reversible by subsequent insulin therapy in vivo. Whether it is the provision of insulin that confers the reversibility or whether a euglycemia provided
Cardiomyopathy
4.57
by other means results in similar findings is not known. The fact that hyperinsulinemia in control animals also seems to affe’ct cardiac actomyosin ATPase suggests that insulin may be specific to the correction. jof the diabetic cardiomyopathic state. The present investigation also confirms the strelptozotocin-induced diabetic cardiomyopathy is a true model of diabetes, since the demonstration of complete reversibility by insulin treatment excludes the possibility of the cardiomyopathy being directly due to strelptozotocin cardiotoxicity. Acknowledgements We wish to thank Gary Ciambrone and Alwyn Murphy for their excellent technical assistance and Mrs Carol Atror for her excellent secretarial assistance. We also wish to thank the Upjohn Co. for their generous donation of streptozotocin. This work was supported by U.S. Public Health Service Grant HL 21482. T. F. Schaible is the recipient of Young Investigator Award HL 25372. This work was presented in part in tlhe Young Investigator Prize Contest, IX World Congress of Cardiology, Moscow, USSR, June 1982.
References 1
2 3 4
5 6 7
8 9 10
BERGMEYER, H. U., BERNT, E., SCHMIDT, F., STORK, H. D-glucose. Determination with hexokiwse and glucose -6-phosphate dehydrogenase. In : Bergmeyer, H. U. (Ed.) Methods of Enzymatic Analysis, pp. I196-1201. New York: Academic Press (1974). BERSOHN, M., SCHEUER, J. Effects of physical training on end-diastolic volume and myocardial performance of isolated rat hearts. Circ Res 40, 510-516 (1977). BERSON, S. A., YALOW, R. S. Insulin in blood and insulin antibodies. Am J Med 40, 676 (1966). BERSON, S. A., YALOW, R. S. General radioimmunoassay. In: Berson, S. A., Yalow, R. S. (Eds) Methods in Investigative and Diagnostic Endocrinology. Part I : General Methodology, pp. 84-120. Amsterdam: North-Holla.nd (1973). BHAN, A. K., SCHEIJER, J. Effects of physical training on cardiac actomyosin adenosine triphosphatase activity. Am J Physiol 223, 1486-1490 (1972). BLEEHEN, N. M., FISHER, R. B. The action of insulin in the isolated rat heart. J Physiol (London) 123, 260-276 (1954). CHAIN, E. B., MANSFORD, K. R. L., OPIE, L. H. Effects of insulin on the pattern of glucose metabolism in the perfused working and Langendorff heart of normal and insulin-deficient rats. Biochem J 115, 537-546 (1969). FACTOR, S. M., BHAN, R., MINASE, T., WOLINSKY, H., SONNENBLICK, E. H. Hypertensive-diabetic cardiiomyopathy in the rat. An experimental model of human disease. Am J Pathol 102, 219-228 (1981). FEIN, F. S., STROBECK, J. E., MALHOTRA, A., SCHEUER, J., SONNENBLICK, E. H. Reversibility of diabetic cardiomyopathy with insulin in rats. Circ Res 49, 1251-1262 (1981). FEIN, F. S., KORNSTEIN, L. B., STROBECK, J. E., CAPASSO, J. M., SONNENBLICK, E. H. Altered myocarclial mechanics in diabetic rats. Circ Res 47, 922-933 (1980).
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Ho~oas~, H. J. L-(.+)-Lactate. Determination with lactic dehydrogenase-and DPN.. In: Bergmeyer, H. U. (Ed.) Methods of Enzymatic Anatysis, pp. 266-270. New York: Academic Press (1963). MALHOTRA, A., PENPARGKUL, S., FEIN, F., SONNENBLICK, E. H., SCHEUER, J. The effect of streptozotocin induced diabetes in rats on cardiac contractile proteins. Circ Res 49, 1243-1250 (1981). MALHOTRA, A., PENPARGKUL, S., SCHAIBLE, T., SCHEUE~, J. Contractile proteins and sarcoplasmic reticulum in physiologic cardiac hypertrophy. Am J Physiol 241, H263-H267 (1981). MILLER, T. B. Cardiac performance of isolated perfused hearts from alloxan diabetic rats. Am J Physiol 236 H808-H812 (1979). MODRAK, J. Collagen metabolism in the myocardium from streptozotocin-diabetic rats. Diabetes 29,547-550 (1980). MURPHY, R. D., VAILAS, A. C., TIPTON, C. M., MATTHES, R. D., EDWARDS, J. G. Influence of streptozotocininduced diabetes and insulin on the functional capacity of rats. J Appl Physiol 50, 482-486 (1981). PAIN, V. M., GARLICK, P. J. Effect of streptozotocin diabetes and insulin treatment on the rate of protein synthesis in tissues of the rat in vim. J Biol Chem 249, 4510-4514 (1974). PENPARGKUL, S., MALHOTRA, A., SCHAIBLE, T., SCHEUER, J. Cardiac contractile proteins and sarcoplasmic reticulum in hearts of rats trained by running. J Appl Physiol 48, 409-413 (1980). PENPARGKUL, S., SCHAIBLE, T., YIPINTSOI, T., SCHEUER, J. The effect of diabetes on performance and metabolism of rat hearts. Circ Res 47, 91 l-921 (1980). PENPARGKUL, S., FEIN, F., SONNENBLICK, E. H., SCHEUER, J. Depressed cardiac sarcoplasmic reticular function from diabetic rats. J Mol Cell Cardiol 13, 303-309 (1981). RANNELS, D. E., HJALMARSON, A. C., MORGAN, G. E. Effects of non-carbohydrate substrates on protein synthesis in muscle. Am J Physiol 226, 538-539 (1974). REGAN, T. J., ETTINGER, P. O., KHAN, M. I., JESRAN, M. U., LYONS, M. M., OLDEWURTEL, H. A., WEBER, M. Altered myocardial function and metabolism in chronic diabetes mellitus without ischemia in dogs. Circ Res 35, 222-237 (1974). REGAN, T. J., LYONS, M. M., AHMED, S. S., LEVINSON, G. E., OLDEWURTEL, H. A., AHMED, M. R., HAIDER, B. Evidnece of cardiomyopathy in familial diabetes mellitus. J Clin Invest 60,885-899 (1978). REGAN, T. J., WV, C. F., YEH, C. K., OLDEWURTEL, H. A., HAIDER, B. Myocardial composition and function in diabetes. The effects of chronic insulin use. Circ Res 49, 1268-1277 (1981). SANDERSON, J. E., BROWN, D. J., RIVELLESE, A., JOHNER, E. Diabetic cardiomyopathy? An echocardiographic study of young diabetes. Br Med J 1, 404-407 (1978). SCHAIBLE, T. F., SCHEIJER, J. Effects of physical training by running or swimming on ventricular performance of rat hearts. J Appl Physiol46, 854-860 (1979). SCHAIBLE, T. F., SCHEUER, J. Cardiac function in hypertrophied hearts from chronically exercised female rats. J Appl Physiol 50, 1140-l 145 (1981). SCHAIBLE, T. F., PENPARGKUL, S., SCHEUER, J. Differences in male and female rats in cardiac conditioning. J App Physiol 50, 112-117 (1981). SCHAIBLE, T. F., MALHOTRA, A., SCHEUER, J. The effect of rat heart size on cardiac performance (Abstract). Fed Proc 41, 1013 (1981). SEGAL, S. A., BLAIR, A. E., WYNGAARDEN, J. B. An enzymatic spectrophotometric method for the determination of pyruvic acid in blood. J Lab Clin Med 48, 137-143 (1956). TAEGTMEYER, H., HEMS, R., KREBS, H. A. Utilization of energy-providing substrates in the isolated working rat heart. Biochem J 186, 701-711 (1980). UMBREIT, W. W., BURRIS, R. H., STAUFFER, J. F. Manornetric techniques (3rd edn) Minneapolis, MN: Burgess, p. 5 (1957). YALOW, R. S., BERSON, S. A. Insulin: radioimmunoassay. In: Berson, S. A., Yalow, R. S. (Eds) Methodsin Part III: Non-pituitary Hormones. pp. 834-870. Amsterdam: Investigative and Diagnostic Endocrinology. North-Holland (1973). YIN, F. C. P., SPURDEON, H. A., WEISFIELDT, M. L., LAKATTA, E. G. Mechanical properties of myocardium from hypertrophied rat hearts. A comparison between hypertrophy induced by senescence and by aortic banding. Circ Res 46, 292-300 (1980). ZAR, J. H. Biostatistical Analysis. Englewood Cliffs, N. J. : Prentice-Hall (1974).