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1 Departments of Pharmacology and Cell Biophysics and 2 Internal Medicine (Division of Cardiology), University of Cincinnati, Cincinnati 45267; 3 Department of Medicine, Case Western Reserve University, Cleveland, Ohio 44106; and 4 Medical Biotechnology Center and Department of Physiology, University of Maryland, Baltimore, Maryland 21201
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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The goal of the study was to determine whether defects in intracellular Ca2+ signaling contribute to cardiomyopathy in streptozotocin (STZ)-induced diabetic rats. Depression in cardiac systolic and diastolic function was traced from live diabetic rats to isolated individual myocytes. The depression in contraction and relaxation in myocytes was found in parallel with depression in the rise and decline of intracellular free Ca2+ concentration ([Ca2+]i). The sarcoplasmic reticulum (SR) Ca2+ store and rates of Ca2+ release and resequestration into SR were depressed in diabetic rat myocytes. The rate of Ca2+ efflux via sarcolemmal Na+/Ca2+ exchanger was also depressed. However, there was no change in the voltage-dependent L-type Ca2+ channel current that triggers Ca2+ release from the SR. The depression in SR function was associated with decreased SR Ca2+-ATPase and ryanodine receptor proteins and increased total and nonphosphorylated phospholamban proteins. The depression of Na+/Ca2+ exchanger activity was associated with a decrease in its protein level. Thus it is concluded that defects in intracellular Ca2+ signaling caused by alteration of expression and function of the proteins that regulate [Ca2+]i contribute to cardiomyopathy in STZ-induced diabetic rats. The increase in phospholamban, decrease in Na+/Ca2+ exchanger, and unchanged L-type Ca2+ channel activity in this model of diabetic cardiomyopathy are distinct from other types of cardiomyopathy.
myocytes; sarcoplasmic reticulum; Na+/Ca2+ exchange
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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APPROXIMATELY 150 million people worldwide suffer from diabetes. In the United States alone, it is estimated that about 16 million people are currently afflicted with diabetes, of which about 1 million have Type 1 diabetes (16). Heart failure is the major cause (~65%) of death among Type 1 diabetic patients (14). Cardiomyopathy has been shown to be a critical factor in heart failure, independent of atherosclerosis, hypertension, and valvular malfunction (12, 32). Cardiomyopathy has been observed even in insulin-treated Type 1 diabetic patients (23). However, the cellular and molecular mechanisms underlying cardiomyopathy and heart failure in Type 1 diabetes are unknown.
Alteration of Ca2+ signaling has been a hallmark of cardiomyopathy and heart failure (29). Changes in critical processes that regulate intracellular Ca2+ concentration ([Ca2+]i), e.g., sarcolemmal L-type Ca2+ channel that triggers Ca2+ release from the sarcoplasmic reticulum (SR) (31), SR Ca2+ release channel (2, 27), Ca2+-(pump)ATPase (SERCA2) (5), dephosphorylation of phospholamban (PLB), which respectively decreases the affinity of SERCA2 for Ca2+ (24), and sarcolemmal Na+/Ca2+ exchanger (NCX), which mediates Ca2+ efflux from the cell (18), have been shown to occur in human cardiomyopathy and failing hearts as well as in many animal models. However, there has not been a thorough examination of these systems in streptozotocin (STZ)-induced diabetic rats to determine whether they contribute significantly to cardiomyopathy in this model.
The goal of the present study was to examine specifically the processes that regulate [Ca2+]i at the cellular and molecular levels to determine whether defective intracellular Ca2+ signaling contributes to cardiomyopathy in STZ-induced diabetic rats. Toward this goal, we traced cardiac contractile dysfunction from live diabetic rats to isolated individual myocytes, then determined whether defects in [Ca2+]i occur in parallel with contractile dysfunction in individual myocytes, and finally determined whether the defects in [Ca2+]i is consistent with alterations of expression and function of proteins that are involved in intracellular Ca2+ signaling. The results of the study demonstrate that defects in [Ca2+]i accompanying contractile dysfunction in STZ-induced diabetic rat heart myocytes. The defects in [Ca2+]i are consistent with alteration of expression and function of its regulatory proteins. The combination of changes in protein expression that alters [Ca2+]i is unlike other types of cardiomyopathy.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Development and characterization of diabetic rats. Six-week-old male Wistar rats, weighing 150 ± 10 g, were made diabetic with STZ as described previously (11). Serum glucose was measured with a glucometer (Bayer; Elkhart, IN), and insulin level was measured using a radioimmunoassay kit (Amersham Life Science; Little Chalfont, Buckinghamshire, UK). Experiments were conducted on 12-wk diabetic rats and age-matched control rats. All the procedures of handling and use of animals were approved by the Institutional Animal Care and Use Committee. The mean blood glucose level of STZ-treated rats was 28.4 ± 1.4 mM (n = 11) compared with 7.2 ± 0.4 mM (n = 11) of the age-matched control rats (n = 11). The mean serum insulin level of the diabetic rats was 0.73 ± 0.21 nM (n = 11) compared with 4.95 ± 0.38 nM (n = 11) of the control rats. These data demonstrate that STZ-treated rats were hyperglycemic and insulin deficient, which are characteristics of Type 1 diabetes.
Measurement of cardiac contractility in vivo by echocardiography. The animals were anesthetized with intraperitoneal injection of pentobarbital sodium (30 mg/kg). M-mode and Doppler echocardiography was conducted as described previously (20).
Measurement of cardiac contractility ex vivo in isolated heart preparation. Cardiac contractility ex vivo was measured in the Langendorff heart preparations perfused with Krebs-Henseleit solution containing 5.5 mM glucose at 37°C and 55 mmHg aortic pressure as described previously (17).
Measurement of cell shortening and [Ca2+]i transients in single cardiac myocytes. Ventricular myocytes were isolated from the hearts of diabetic and age-matched control rats, and cell shortening and [Ca2+]i with fura 2 fluorescence were measured as described previously (28). SR Ca2+ content, kinetics of SR Ca2+ uptake and release, and Ca2+ efflux via NCX were estimated according to a published procedure (3). The raw data from Felix software (Photon Technology International; Monmouth, NJ) was transported to IonWizard software (IonOptix; Milton, MA). The IonWizard program provided data in measured parameters from 10 selected consecutive contractions and corresponding [Ca2+]i transients. Whereas the measured levels of [Ca2+]i may vary with different indicators and methods used in different laboratories, it is assumed the method employed in this study should provide an accurate comparison in the relative level between normal and diabetic rat myocytes.
Measurements of L-type Ca2+ channel activity and [Ca2+]i by confocal microscopy. Ventricular myocytes were isolated and stored at room temperature (22-25°C) in Dulbecco's modified Eagle's medium (Sigma Chemical; St. Louis, MO) (15). An Axopatch-200A or Axopatch-200B amplifier (Axon Instruments) was used to patch-clamp single myocytes (whole cell configuration) and measure membrane currents. Confocal microscopy was used to measure and image [Ca2+]i with fluo-3 (33).
Measurements of protein and phosphorylated PLB levels.
Quantitative immunoblot was used to determine individual protein levels
and phosphorylated PLB level (25). The antibodies used
were PLB (1:1,000, Affinity Bioreagents; Golden, CO), calsequestrin (CSQ, 1:5,000, a gift from Dr. Larry Jones, Indiana University; Indianapolis, IN), NCX (1:500, Affinity Bioreagents), sarcomeric 
-actin (1:2,000, Sigma Chemical), SERCA2 (1:400, Santa Cruz
Biotechnology; Santa Cruz, CA), RyR (1:700, Affinity Bioreagents), and
phosphorylated PLB at serine-16 and threonine-17 (1:5,000,
Fluorescience; Leeds, UK), with the appropriate secondary antibodies
conjugated to horseradish peroxidase.
Measurement of Ca2+ uptake into SR. The initial rate of Ca2+ uptake into SR was determined by the modified Millipore filtration technique, with varying free [Ca2+] (26), in the presence of 1 µM cAMP-dependent protein kinase inhibitor (Sigma Chemical), 0.8 µM Ca2+-calmodulin-dependent protein kinase inhibitor (Upstate Biotechnology; Lake Placid, NY), and 1 µM phosphatases inhibitor calyculin A (Upstate Biotechnology).
Data and statistical analyses. Electrophysiological data were analyzed using combinations of pCLAMP 6.01 or 8.0 (Axon Instruments), IDL (RSI; Boulder CO), Excel (Microsoft), and Origin (v.6) software. All data are expressed as means ± SE. Two-sample comparisons were performed using Student's t-test. For all analyses P < 0.05 was considered significant.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Cardiac contractile dysfunction in vivo.
Doppler and M-mode echocardiography provides information on cardiac
dimension and contractile function in vivo. Therefore, echocardiography
was employed to determine the pattern and the extent of cardiac
contractile dysfunction in vivo in diabetic rats. The heart rate (HR)
in diabetic rats (264 ± 15 beats/min; n = 6) was
significantly lower than that of the control rats (346 ± 8 beats/min; n = 6). There was no significant change in
left ventricular (LV) chamber dimension as indicated by the unchanged LV end-diastolic dimension in diabetic rats (6.86 ± 0.27 mm)
compared with that of control rats (6.73 ± 0.30 mm) and the
unchanged LV end-systolic dimension in diabetic rats (2.86 ± 0.17 mm) compared with the control rats (2.52 ± 0.23 mm). The LV peak
ejection rate (PER) and peak-filling rate (PFR) were significantly
lower, respectively, by 34% (Fig.
1A) and 28% (Fig.
1B) in diabetic rats compared with control rats. The LV
ejection time and isovolumic relaxation time in diabetic rats were
significantly longer by 69% (Fig. 1D) and 80% (Fig.
1E), respectively. The LV circumferential shortening velocity (Vcf) in diabetic rats was decreased
significantly by 42% (Fig. 1C). Because
Vcf is an index normalized by HR
(20), the decreased level in diabetic rats indicates that
LV contractile dysfunction is not due to decreased HR. However, there
was no significant decrease in fractional shortening in diabetic rat hearts (50 ± 5%) compared with control rat hearts (48 ± 2%), indicating that there is no heart failure the diabetic rats.
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Cardiac contractile dysfunction ex vivo. The contractile dysfunction observed in vivo could be partly due to extrinsic factors, such as changes in circulating metabolites or hormones. In isolated heart preparations, the influence of extrinsic factors is eliminated, which allows for the evaluation of intrinsic contractile dysfunction. Therefore, contractile function was examined ex vivo in isolated heart preparations.
The basal HR of diabetic rats (185 ± 13 beats/min; n = 4) was significantly lower than that of control rats (255 ± 8 beats/min; n = 4). The LV rate of development of systolic pressure and the rate of decline of the pressure were lower, respectively, by 29% (Fig. 2A) and 22% (Fig. 2B) in diabetic rat hearts. In diabetic rat hearts, time to peak pressure (TPP) was longer by 28% (Fig. 2C) and time to half-relaxation (RT50) from the peak pressure was longer by 71% (Fig. 2D). Coronary resistance in diabetic rat hearts was significantly higher by 73%. A small (9%) decrease in LV intraventricular peak pressure was observed in diabetic rat hearts but was found statistically insignificant. There was no increase in LV end-diastolic pressure in diabetic rat hearts, indicating the absence of heart failure.
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Contractile dysfunction in isolated single myocytes. The changes observed in intact hearts ex vivo could be due to decreased myocardial perfusion. When examined under identical perfusion conditions, this factor is eliminated in isolated myocytes. Therefore, contractile function was examined in isolated myocytes.
The fraction of viable myocytes, in terms of rod-shape and Ca2+-tolerant cells, isolated from control (69 ± 7%) and diabetic rat hearts (60 ± 5%) was not significantly different. Representative tracings of contraction transients of a single myocyte from a control rat heart and a single myocyte from a diabetic rat heart that were separately stimulated at 0.2 Hz are shown in Fig. 3A. The rates of contraction (+dL/dt) and relaxation (
dL/dt) in diabetic rat myocytes were 65%
lower than that of control rat myocytes. In diabetic rat myocytes, the
amplitude of contraction was 46% lower and TTP and RT50
were 52% longer than that of control rat myocytes. The 
of
relaxation was about 89% longer in diabetic rat myocytes than control
rat myocytes.
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Changes in [Ca2+]i
cycling in isolated single myocytes.
To determine whether contractile dysfunction observed in isolated
single myocytes from diabetic rat hearts is due to altered intracellular Ca2+ homeostasis,
[Ca2+]i transient was measured simultaneously
with contraction transients described above. The basal
[Ca2+]i level before electrical stimulation
was similar between the control (51 ± 4 nM, n = 12 hearts) and diabetic rat myocytes (50 ± 6 nM,
n = 10 hearts). Representative
[Ca2+]i transients of a myocyte of a control
rat heart and a myocyte of a diabetic rat heart that were stimulated at
0.2 Hz are shown in Fig. 3A (bottom). The
diastolic [Ca2+]i was 44 ± 3 nM
(n = 12 hearts) in control rat myocytes and was 48 ± 6 nM (n = 10 hearts) in diabetic rat myocytes at 0.2 Hz. The cumulative kinetic data of [Ca2+]i
transients corresponding to the cell shortening at 0.2 Hz is presented
in Fig. 3C. The rate of rise of
[Ca2+]i level
(+d[Ca2+]/dt) was 74% lower, the rate of
decline (d[Ca2+]/dt) was 77% lower, and the
amplitude of [Ca2+]i was 71% lower in
diabetic rat myocytes than that in control rat myocytes. The TTP
[Ca2+]i was 49% longer and the
RT50 decline in [Ca2+]i was 50%
longer in diabetic rat myocytes. The of rate of
[Ca2+]i decline was 70% longer in diabetic
rat myocytes.
d[Ca2+]/dt indicates that the rate of
Ca2+ resequestration into SR may be decreased. However,
decreased Ca2+ efflux via NCX may also contribute to a
decreased rate of [Ca2+]i decline because it
is also involved, albeit modestly, in this process (3,
30).
Evaluation of SR Ca2+ sequestration and Ca2+ efflux via NCX in situ in isolated single myocytes. To determine whether depression of SR and NCX contributes to defects in [Ca2+]i cycling, the function of these systems was determined in situ in isolated single myocytes. The rates of Ca2+ release and sequestration into SR, the magnitude of SR Ca2+ store, and the rate of Ca2+ efflux via NCX were determined in isolated myocytes by comparing the rate of rise, the amplitude, and the rate of [Ca2+]i transient after induction of Ca2+ release from SR by caffeine in normal Krebs-Henseleit solution and in Na+- and Ca2+-free Krebs-Henseleit solution (3).
Representative records of caffeine-induced [Ca2+]i transients after 30 s rest from 0.2 Hz stimulation are presented in Fig. 4A. Cumulative data of the amplitude and kinetics of [Ca2+]i transient decline after 10 mM caffeine-induced Ca2+ release from SR are presented as an inset table in Fig. 4. The amplitude of [Ca2+]i transients induced by caffeine in diabetic rat myocytes was 59% lower than that of control rat myocytes, which indicates that the amount of Ca2+ stored in SR in diabetic rat myocytes was significantly lower. The rate of rise of [Ca2+]i was decreased by 71%, and TTP [Ca2+]i was prolonged by 64% in diabetic rat myocytes, which indicate defects in Ca2+ release from SR in diabetic rat myocytes. The rate of [Ca2+]i decline was decreased by 73%, and the RT50 and of the
rate of decline were prolonged by 67% and 100%, respectively, in
diabetic rat myocytes. The rate of Ca2+ sequestration into
SR, calculated by subtracting the rate of [Ca2+]i decline in the presence of caffeine
from that after stimulation at 0.2 Hz, was significantly
(P < 0.05) lower in diabetic rat myocytes (0.482 ± 0.125 µM/s; n = 8) compared with control rat myocytes (0.149 ± 0.50 µM/s; n = 8). The of
the rate of [Ca2+]i decline after 0.2 Hz
stimulation and caffeine application in normal Na and Ca containing
medium and in Na+-free/Ca2+-free (0Na0Ca)
medium are presented in Fig. 4B. Because the rate of
[Ca2+]i decline in the presence of caffeine
attributed to Ca2+ efflux via NCX, sarcolemmal (SL)
Ca2+ pump, and Ca2+ uptake into mitochondria
(3, 30), prolongation of in diabetic rat myocytes in
the presence of caffeine indicates a decrease in activity of one or
more of these systems. The rate of [Ca2+]i
decline after caffeine-induced [Ca2+]i
transient in 0Na0Ca medium has been attributed to Ca2+
efflux via sarcolemmal Ca2+ pump and mitochondrial
Ca2+ uptake (3, 30). The of
[Ca2+]i decline in diabetic rat myocytes
under these conditions was similar to that of control rat myocytes,
which indicates that the prolongation of in the presence of
caffeine in Na- and Ca-containing medium is due to a decrease in NCX
activity.
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Voltage-gated L-type Ca2+ channel
current activity and imaging of
[Ca2+]i transient with
confocal microscopy.
To determine whether a decrease in L-type Ca2+ channel
activity contributes to the decreased rate of Ca2+ release
from the SR in diabetic rat heart myocytes, L-type Ca2+
channel activity was examined with whole cell patch-clamp technique. Single myocytes isolated from diabetic rat hearts depolarized from 40
to +60 mV exhibited smaller [Ca2+]i
transients compared with those from age-matched control rats (cf. Fig.
5, A with B). The
magnitude and the rate of decay of [Ca2+]i
were attenuated in diabetic rat myocytes. The L-type Ca2+
current (ICa) density (pA/pF) was similar at all
voltages from 40 to +60 mV (Fig. 5C). However, peak
[Ca2+]i transients were significantly smaller
in diabetic rat myocytes (Fig. 5D). The isochronal
[Ca2+]i transient decay (200 ms after peak)
at 0 mV was also significantly decreased (Fig. 5E). The
membrane capacitance was significantly smaller (Fig. 5F),
indicating that diabetic rat myocytes are smaller in size.
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Changes in SR proteins expression. To determine whether decreased SR function observed in diabetic rat myocytes is due to decreased expression of SR Ca2+ transport proteins, SERCA2, total and phosphorylated PLB, RyR, and CSQ (SR luminal Ca-buffering protein) levels were measured by the quantitative immunoblot technique.
The level of SERCA2 protein in diabetic rat hearts was decreased by 30% compared with control rat hearts (Fig. 6). The total PLB protein level in diabetic rat hearts was increased by 150% compared with control rat hearts (Fig. 6). The PLB-to-SERCA2 ratio in control rat hearts was 0.94 ± 0.09 (n = 4) and in diabetic rat hearts was 3.33 ± 0.59 (n = 4). This produces a 3.5-fold increase in the PLB-to-SERCA2 ratio in diabetic rat hearts. The basal levels of phosphorylated PLB at serine-16 and threonine-17 were also determined. The phosphoserine level was significantly decreased by 54% in diabetic rat hearts (Fig. 6). The phosphothreonine level was decreased by 70% in diabetic rat hearts. The decreased phosphorylation of PLB indicates an increased nonphosphorylated PLB level because the total PLB protein level is increased in the diabetic rat hearts. The protein level of RyR was significantly decreased by 37% in diabetic rat hearts (Fig. 6). The CSQ protein level in diabetic rat hearts was not significantly different from that of the control rat hearts (Fig. 6).
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Changes in NCX and -actin protein expression.
To determine whether decreased NCX function observed in diabetic
rat myocytes is due to its decreased protein level, its protein level was measured by the quantitative immunoblot technique. To determine whether the changes observed in SR and NCX proteins levels
are part of a general decrease in protein expression in diabetic rat
hearts, the levels of -actin protein, which is not structurally and
functionally associated with SR or NCX, was also determined. The NCX
protein level in diabetic rat hearts was decreased by 45% compared
with that of control rat hearts (Fig. 6). However, there was no
significant change in the -actin protein level in diabetic rat
hearts (Fig. 6).
-actin and CSQ protein levels
and the increase in PLB protein level indicate that the changes in NCX
and SR Ca2+ transport proteins are selective and are not
part of a general decline in protein expression in diabetic rat hearts.
Ca2+ uptake into SR in vitro.
Because SERCA2 protein level is decreased and nonphosphorylated PLB
level and PLB-to-SERCA2 ratio are increased in diabetic rats hearts,
the Vmax and apparent affinity of the SR
Ca2+ pump for Ca2+ was determined in
vitro. The results presented in Fig.
7 demonstrate that the concentration of
free Ca2+ required for half of the maximum rate of
Ca2+ uptake (EC50) was increased by 106% and
that the Vmax of Ca2+ uptake into SR
of diabetic rat hearts was decreased by 39% compared with that of
control rat hearts.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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In this study, contractile dysfunction was traced from intact animals to single myocytes and demonstrated that intrinsic defects in intracellular Ca2+ signaling contribute to cardiomyopathy in STZ-induced Type 1 diabetes. A major strength of this study is that parallel defects in contractile function were identified at three different levels of complexity, i.e., live animals, isolated hearts, and isolated myocytes. Moreover, parallel defects in contraction and [Ca2+]i transients in the same myocyte clearly indicate that defective intracellular Ca2+ signaling contributes to contractile dysfunction. Selective alteration of expression and function of SR and NCX proteins underscores the defects in [Ca2+]i and contraction in diabetic rat myocytes. However, there has been controversy regarding alteration of [Ca2+]i in myocytes isolated from STZ-induced diabetic rat hearts (19, 37). Unlike the present study, the critical systems that regulate [Ca2+]i were not examined in these studies to verify the findings. The present study demonstrates the defects in [Ca2+]i cycling corroborated by alteration of the expression and function of the proteins that regulate it.
The reduction of the amplitude and kinetics of
[Ca2+]i transient in diabetic rat myocytes
observed in this study is due to a reduction of the ability of the SR
to sequester Ca2+. The direct evidence in favor of this
conclusion is provided by the following observations: 1) a
reduction of the SR Ca2+-ATPase protein and its activity
that pumps Ca2+ into SR, 2) an increment of the
PLB protein with a reduction in its phosphorylation decreases the
affinity for Ca2+ and the activity of the SR
Ca2+ pump, and 3) a reduction of the SR
Ca2+ content. Further indirect support for this conclusion
comes from the observation that the level of trigger Ca2+
that enters through the voltage-dependent Ca2+ channel, as
measured by ICa density, is unchanged in
diabetic rat myocytes. A decrease in its activity could have altered
[Ca2+]i transient kinetics. Moreover, absence
of any alteration in the geometry of the organization of the EC
coupling system in this model (see below) indicates that the current
density of ICa did not alter the
"activation" component of EC coupling by altering the dominant
actions of local subcellular [Ca2+]i on the
triggering of SR Ca2+ release (4). Whereas the
features discussed above could not account for the reduction in
[Ca2+]i transient, shortening the action
potential duration could have; but the action potential is not reduced
in this model (34). Further evidence that action potential
duration is not a contributing factor in the reduction of
[Ca2+]i transient comes from the observation
that the [Ca2+]i transient was still
diminished when the duration of waveform controlled the depolarization
in the patch-clamp experiments (Fig. 5). The reduction of NCX
expression and function would also contribute, albeit in a minor way,
to the reduction in the rate of SR Ca2+ uptake and the rate
of decline of [Ca2+]i. Finally, the absence
of any significant change in the resting [Ca2+]i level suggests that the overall
sarcolemmal "pump-leak balance" is largely unchanged in this model.
Our results indicate that reduction in the intracellular
Ca2+ signaling would be sufficient to account for the
reduction in the cardiac contractility in this model. Improvement of
cardiac contractility by overexpression of SERCA2 in STZ-induced
diabetic mice (9) underscores the role of defective
[Ca2+]i in diabetic cardiomyopathy. However,
it is worth noting that additional factors, such as alteration of
myofilament proteins and Ca2+ sensitivity (1)
or protein kinase C
2 overexpression (21), may also contribute to the reduced contractility on this model.
The intrinsic defects in myocytes in this model of Type 1 diabetes are congruent with the clinical findings of the existence of cardiomyopathy independent of atherosclerosis, vascular, or valvular diseases in human Type 1 diabetes (12, 23, 32). Thus we report here clinically significant cardiac contractile dysfunction in the STZ-induced model of Type 1 diabetes. We deduce that the cardiac muscle defect is due in part to the metabolic alterations that occur in the near absence of insulin secretion (~85% reduction of serum insulin level). Furthermore, we deduce that changes in the cellular expression of specific proteins are likely to occur in the STZ-induced diabetic rats because insulin, in addition to affecting the metabolism of glucose and lipids, also influences gene expression (36). On the other hand, hypothyroidism caused by diabetes may also contribute to cardiac gene expression (8). At this point in our study, we do not know exactly which genes may be directly or indirectly affected by insulin deficiency and the absence of regular fluctuation of insulin levels. Future studies will be needed to better identify and characterize these features.
As has been reported in many models of cardiomyopathy and heart failure, the nature of the cardiac dysfunction observed in the experiments presented here involves reduced contractile function of the heart and of the myocytes. Features such as decrease in SERCA2 and RyR and decrease in [Ca2+]i and contraction transient are similar to observations made in other models of cardiomyopathy or heart failure. However, in some features it is distinct from other types of cardiomyopathy. In the diabetic rat hearts there is a reduction of the cellular [Ca2+]i transient in the absence of significant change in ICa density even with reduction of expression and function of the SR Ca2+ ATPase. Both of these elements have been reported decreased in forms of heart failure attributed to many causes, including pressure overload (15), viral myocarditis, muscle LIM protein knockout (35), and myocardial infarction (10). There are also other distinctive features of cardiac contractile dysfunction in this model of Type 1 diabetes. First, whereas there is no heart failure phenotype, there is significant systolic and diastolic dysfunction. Second, there appears to be no cellular hypertrophy. Indeed, the cell size is reduced by about 35% as measured by cellular capacitance. Whereas this could reflect the general wasting syndrome associated with untreated Type 1 diabetes, the overall response is not simply that of general downregulation of protein synthesis. The third distinctive feature of this cardiomyopathy, the overexpression of PLB protein, provides evidence against this notion. Importantly, this particular result of the study indicates that proteins are specifically up or downregulated in Type 1 diabetes and that this disease is not simply a manifestation of a global muscle-wasting syndrome. A recent report (22) suggests that targeted overexpression of PLB protein can cause cardiac contractile dysfunction. On the other hand, cardiac hyperperformance is observed on PLB gene knockout (26). In the diabetic rat hearts, not only there is an increase in total PLB protein but also there is a reduction in the fraction of PLB that is phosphorylated at both serine-16 and threonine-17. This observation highlights a fourth distinctive feature, i.e., PLB is not hyperphosphorylated in this model unlike the PLB overexpression model (6). The decreased PLB phosphorylation is consistent with not only the decreased function of SR but also with the absence of a hyperadrenergic state that is indicated by the slower HR in diabetic rats in this study and data from others (13). Finally, a fifth distinctive feature of this model is that there are reductions of RyR and NCX proteins that regulate [Ca2+]i, which have been shown unchanged or increased in other models (2, 18, 27). Thus the results of the study clearly demonstrate a pattern of molecular changes that are distinct from other types of cardiomyopathy but are consistent with the observed defects in [Ca2+]i and contractile function.
In summary, we have demonstrated cardiac contractile dysfunction at three levels of complexities that occurs in STZ-induced Type 1 diabetic rats. Significant systolic and diastolic dysfunction that can be traced to cellular and molecular levels occurs before overt heart failure develops. It is caused by primary defects in intracellular Ca2+ signaling that expectedly attenuates [Ca2+]i transients and that contributes to poor contractile performance. The cardiomyopathy in this model of diabetes is similar in some aspects to nondiabetic cardiomyopathies. However, there are features such as increase in PLB, decrease in phosphorylated fraction despite the increase in total PLB, decrease in NCX, and unchanged L-type Ca2+ channel activity that are distinct from other types of cardiomyopathy.
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ACKNOWLEDGEMENTS |
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We thank Jianhua Zhang, Gilbert Newman, and Ali Tsurov for technical assistance in some of the experiments and Dr. Evangelia Kranias and Ashley Mattingly for critically reading the paper.
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FOOTNOTES |
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This work was supported by grants from the National Heart, Lung, and Blood Institute (R01-HL56782) and American Diabetes Association. Kin Man Choi is a recipient of Albert Ryan Fellowship.
Address for reprint requests and other correspondence: M. A. Matlib, Dept. of Pharmacology and Cell Biophysics, College of Medicine, Univ. of Cincinnati, 231 Albert B. Sabin Way, PO Box 670575, Cincinnati, OH 45267-0575 (E-mail: matlibma{at}uc.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
June 27, 2002;10.1152/ajpheart.00313.2002
Received 25 February 2002; accepted in final form 24 June 2002.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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0.9). Slope of the
regression line of the control rat was taken as 100% to determine the
percent change in the diabetic rat hearts. Open
bars, data from control rat hearts (n = 4);
closed bars, data from diabetic rat hearts
(n = 4). Data are means ± SE. *P < 0.05 vs. control rats.
