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Am J Physiol Heart Circ Physiol 283: H1398–H1408, 2002. First published June 27, 2002; 10.1152/ajpheart.00313.2002. Defective intracellular Ca2⫹ signaling contributes to cardiomyopathy in Type 1 diabetic rats KIN M. CHOI,1 YAN ZHONG,1 BRIAN D. HOIT,2,3 INGRID L. GRUPP,1 HARVEY HAHN,2 KEITH W. DILLY,4 SILVIA GUATIMOSIM,4 W. JONATHAN LEDERER,4 AND MOHAMMED A. MATLIB1 1 Departments of Pharmacology and Cell Biophysics and 2Internal Medicine (Division of Cardiology), University of Cincinnati, Cincinnati 45267; 3Department of Medicine, Case Western Reserve University, Cleveland, Ohio 44106; and 4Medical Biotechnology Center and Department of Physiology, University of Maryland, Baltimore, Maryland 21201 Received 25 February 2002; accepted in final form 24 June 2002 Choi, Kin M., Yan Zhong, Brian D. Hoit, Ingrid L. Grupp, Harvey Hahn, Keith W. Dilly, Silvia Guatimosim, W. Jonathan Lederer, and Mohammed A. Matlib. Defective intracellular Ca2⫹ signaling contributes to cardiomyopathy in Type 1 diabetic rats. Am J Physiol Heart Circ Physiol 283: H1398–H1408, 2002. First published June 27, 2002; 10.1152/ajpheart.00313.2002.—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. 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). Cardio- myopathy 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 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: [email protected]). 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. myocytes; sarcoplasmic reticulum; Na⫹/Ca2⫹ exchange H1398 0363-6135/02 $5.00 Copyright © 2002 the American Physiological Society http://www.ajpheart.org DIABETIC CARDIOMYOPATHY 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. MATERIALS AND METHODS Development and characterization of diabetic rats. Sixweek-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 AJP-Heart Circ Physiol • VOL H1399 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 M cAMP-dependent protein kinase inhibitor (Sigma Chemical), 0.8 M Ca2⫹-calmodulin-dependent protein kinase inhibitor (Upstate Biotechnology; Lake Placid, NY), and 1 M 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. RESULTS 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. Thus echocardiography demonstrates significant LV systolic and diastolic dysfunctions in diabetic rat hearts in vivo without any LV chamber dilation or sign of heart failure. The unchanged cardiac dimension observed in this study is consistent with results of Depre et al. (7). However, cardiac functional data were not presented in that study and therefore could not be compared. 283 • OCTOBER 2002 • www.ajpheart.org H1400 DIABETIC CARDIOMYOPATHY Fig. 1. Contractile dysfunction in vivo determined by echocardiography. Top, representative M-mode echocardiograms of a control rat and a diabetic rat. Bottom, cumulative data of peak ejection rate (PER, A), peak filing rate (PFR, B), circumferential shortening velocity (Vcf, C), ejection time (ET, D), and isovolumic relaxation time (IVRT, E). Open bars, data from control rats (n ⫽ 6 rats); closed bars, data from diabetic rats (n ⫽ 6 rats). Data are means ⫾ SE of each group. *P ⬍ 0.05 vs. control rats. Circ, circumferential dimension. 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. The results demonstrate that systolic and diastolic dysfunction observed in vivo by echocardiography is largely preserved ex vivo in isolated heart preparations. Thus it appears that cardiac contractile dysfunction in diabetic rats is mainly due to intrinsic changes within the heart, although the conditions that exist in vivo, particularly with respect to substrates, were not maintained ex vivo. AJP-Heart Circ Physiol • VOL 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 rodshape 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. The results demonstrate significant contractile dysfunction in isolated myocytes in parallel with systolic and diastolic dysfunction observed in diabetic rat hearts in vivo and ex vivo with the exception of a significant decrease in the amplitude of shortening. However, the magnitude of changes is greater in isolated myocytes compared with those in intact hearts in vivo and ex vivo. Two factors that could be responsible for this disparity are lack of external resistance and similarity in rate of stimulation of myocytes unlike 283 • OCTOBER 2002 • www.ajpheart.org H1401 DIABETIC CARDIOMYOPATHY Fig. 2. Contractile dysfunction ex vivo in Langendorff-perfused heart preparations. Top, typical tracings of left ventricular (LV) intraventricular pressure development and contractile kinetics from a control rat and a diabetic rat. Cumulative results of rate of pressure development (⫹dP/dt) and decline (⫺dP/dt), time to peak pressure (TPP), and time to half relaxation (RT50) are shown in A, B, C, and D, respectively. Open bars, data from control rats (n ⫽ 4 hearts); closed bars, data from diabetic rats (n ⫽ 4 hearts). Data are means ⫾ SE of each group. *P ⬍ 0.05 vs. control rats. intact hearts in vivo and ex vivo in which there were differences in HR between control and diabetic rats. 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. The results demonstrate that changes in [Ca2⫹]i are associated with parallel changes in contraction-relaxAJP-Heart Circ Physiol • VOL ation in single myocytes from diabetic rats. Thus results also indicate that defects in [Ca2⫹]i cycling contribute to the defects in contraction-relaxation in individual myocytes of diabetic rat hearts. The decreased ⫹d[Ca2⫹]/dt indicates that the rate of SR Ca2⫹ release and/or L-type of Ca2⫹ channel activity, which triggers Ca2⫹ release from SR may be decreased. The decrease in amplitude of [Ca2⫹]i indicates that the SR Ca2⫹ store may be decreased. The decreased ⫺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 283 • OCTOBER 2002 • www.ajpheart.org H1402 DIABETIC CARDIOMYOPATHY Fig. 3. Cell shortening and intracellular Ca2⫹ concentration ([Ca2⫹ ]i) transients in isolated cardiomyocytes. A: representative records of cell shortening and [Ca2⫹]i transient of a control and a diabetic rat myocyte. Shortening was measured using a cell-edge detection system described under MATERIALS AND METHODS. [Ca2⫹]i transient and cell shortening were recorded simultaneously with electrical field stimulation at 0.2 Hz. B: cumulative data presented in bar graphs of rate of contraction development (⫹dL/dt) and decline (⫺dL/dt), percent cell shortening, TTP, RT50, and . C: cumulative data of rate of [Ca2⫹] development (⫹d[Ca2⫹]/dt) and decline (⫺d[Ca2⫹]/ dt), the amplitude of Ca2⫹ transients, TTP, RT50, and of ⫺d[Ca2⫹]/dt. Open bars, data from control rats; closed bars, data from diabetic rats. Data are means ⫾ SE of from 10 rat hearts from each group. *P ⬍ 0.05 vs. control rats. 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) AJP-Heart Circ Physiol • VOL lower in diabetic rat myocytes (0.482 ⫾ 0.125 M/s; n ⫽ 8) compared with control rat myocytes (0.149 ⫾ 0.50 M/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 prolon- 283 • OCTOBER 2002 • www.ajpheart.org DIABETIC CARDIOMYOPATHY H1403 Fig. 4. Caffeine-induced [Ca2⫹]i transients in cardiomyocytes. A: representative tracings of caffeine-induced [Ca2⫹]i transients in normal Krebs-Henseleit (KH) of a control and a diabetic rat myocyte following a 30-s rest after 30-s pacing at 0.2 Hz are shown. Arrows indicated point of 10 mM caffeine addition. There was a 1.0- to 1.5-s mixing time delay of response to caffeine addition. Caffeine was washed out, and the cells were again stimulated periodically until contraction and Ca2⫹ transients restored to precaffeine levels. Inset, kinetic data of caffeine-induced [Ca2⫹]i. B: of the rate of [Ca2⫹]i decline after stimulation at 0.2 Hz and after caffeine application in normal Na and Ca containing KH medium and in ONa0Ca KH medium. Open bars, data from control rat heart myocytes; closed bars, data from diabetic rat heart myocytes. Data are means ⫾ SE of each group (n ⫽ 6 control rats; n ⫽ 4 diabetic rats). *P ⬍ 0.05 vs. control rats. gation of in the presence of caffeine in Na- and Ca-containing medium is due to a decrease in NCX activity. The results demonstrate that the magnitude of SR Ca2⫹ store, the rates of Ca2⫹ release and resequestration into SR, and Ca2⫹ efflux via NCX are significantly decreased and largely contribute to the defects in [Ca2⫹]i transients in myocytes of diabetic rats. The decreased rate of Ca2⫹ release from the SR could be due to decreased activity of the RyR and/or the sarcolemmal voltage-gated L-type Ca2⫹ channel. The decreased function of SR and NCX in regulating [Ca2⫹]i myocytes from diabetic rat hearts could be due to decreased expression of SR Ca2⫹ transport and NCX proteins. 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 AJP-Heart Circ Physiol • VOL 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. The results demonstrate that L-type Ca2⫹ channel function is normal in diabetic rat heart myocytes even though there is a significant decrease in [Ca2⫹]i cycling. Thus it may be concluded that decreased function of the RyR and a decreased SR Ca2⫹ store may be responsible for the decreased rate of Ca2⫹ release from SR. 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) 283 • OCTOBER 2002 • www.ajpheart.org H1404 DIABETIC CARDIOMYOPATHY Fig. 5. Voltage-gated L-type Ca2⫹ channel activity and simultaneous imaging of [Ca2⫹]i by confocal microscopy. Depolarization-activated [Ca2⫹]i transients and membrane currents were examined in rat ventricular myocytes using singlecell patch-clamp methods and confocal imaging of [Ca2⫹]i. Representative data from a control (A) and a diabetic ventricular myocyte (B) are shown during a depolarization from ⫺80 to 0 mV for 200 ms. Protocol involves first slowly (0.06 mV/ms) depolarizing the cell from ⫺80 to ⫺50 mV and holding the potential at ⫺50 mV for 50 ms to inactivate Na⫹ current and T-type Ca2⫹ current before depolarizing the cell to 0 mV for 200 ms. Top traces show a diagram of the voltage step from ⫺50 to 0 mV and the repolarization to ⫺80 mV. The other components of each panel are (from top to bottom) the [Ca2⫹]i transient (as fractional fluorescence F/F0), the line scan image of [Ca2⫹]i, and the L-type Ca2⫹ current (ICa,L) density (pA/pF). C: voltage dependence of ICa,L, plotted as current density (pA/pF) obtained for test potentials from ⫺40 to ⫹60 mV. No significant difference was observed between ICa,L from control (n ⫽ 5) and diabetic (n ⫽ 21) myocytes. D: voltage dependence of [Ca2⫹]i transient (measured as F/F0) in control (n ⫽ 5) and diabetic (n ⫽ 16) ventricular myocytes. E: isochronal (200 ms after peak) percent decay of [Ca2⫹]i transient at 0 mV measured in control (n ⫽ 6) and diabetic (n ⫽ 17) ventricular myocytes. F: membrane capacitance (pF) measured in control (n ⫽ 10) and diabetic (n ⫽ 21) ventricular myocytes. Bar graph values are the following. Membrane capacitance (pF): control 173.82 ⫾ 8.38 (n ⫽ 10), diabetic 112.71 ⫾ 5.66 (n ⫽ 21), P ⬍ 0.001. Isochronal % [Ca2⫹]i transient decay at 0 mV: control 37.04 ⫾ 1.31 (n ⫽ 6), diabetic 18.88 ⫾ 1.29 (n ⫽ 17), P ⬍ 0.001. *P ⬍ 0.05 vs. control rats. 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 AJP-Heart Circ Physiol • VOL 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). The decreased RyR protein level with normal L-type Ca2⫹ channel function indicates that decreased RyR function is responsible for the slow release of Ca2⫹ from SR and prolongation of time to peak [Ca2⫹]i transients observed in diabetic rat myocytes. The decreased SERCA2 protein level, increased nonphosphorylated PLB population, and increased PLB-to-SERCA2 ratio indicate that decreased SERCA2 function is responsible for the slow rate of Ca2⫹ sequestration into the SR in diabetic rat myocytes. The decreased SERCA2 level should lower the maximum velocity of SR Ca2⫹ pump, 283 • OCTOBER 2002 • www.ajpheart.org H1405 DIABETIC CARDIOMYOPATHY Fig. 6. Quantitative immuoblots of sarcoplasmic reticulum (SR) and Na⫹/ Ca2⫹ exchanger (NCX) protein expressions. Left, representative quantitative immunoblots of Sr Ca2⫹-ATPase (SERCA2a), phospholamban (PLB), ryandine receptor (RyR), calsequestrin (CSQ), ␣-actin, NCX, PLB phosphoserine-16 (PS-16), and PLB phosphothreonine-17 (PT-17). Density of the bands of each protein with increasing amounts of homogenate protein were plotted to obtain a linear regression line (r2 ⱖ 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. and the increased nonphosphorylated PLB should lower the affinity of the Ca2⫹ pump for Ca2⫹ (24). The unchanged CSQ protein level indicates that the decrease in SR Ca2⫹ store observed in diabetic rat myocytes is not due to a change in the SR luminal Ca2⫹buffering protein level. The magnitude of SERCA2 and RyR protein levels observed in 12-wk diabetic rats is similar to that observed in 6-wk diabetic rats (38). However, the magnitude of the PLB protein level in 12-wk diabetic rats is increased by about 150% compared with about 60% in 6-wk diabetic rats. This indicates that among SR proteins, only the PLB level increases with the duration of diabetes. AJP-Heart Circ Physiol • VOL 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 283 • OCTOBER 2002 • www.ajpheart.org H1406 DIABETIC CARDIOMYOPATHY change in the ␣-actin protein level in diabetic rat hearts (Fig. 6). The decrease in NCX protein level indicates that it is responsible for the decrease of NCX function observed in situ in individual myocytes of diabetic rats. The lack of change in ␣-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. The increased EC50 of Ca2⫹ uptake indicates a decrease in the apparent affinity of SR Ca2⫹ pump for Ca2⫹, which is consistent with the increased nonphosphorylated PLB protein level; likewise the decreased Vmax is consistent with decrease in SERCA2 protein level. Thus these results demonstrate that the alteration of expression of SR proteins changed its function in diabetic rat hearts. DISCUSSION 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 dys- function. 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 experi- Fig. 7. Ca2⫹ uptake into cardiac SR of cardiac of control and diabetic rats. Initial linear rate of Ca2⫹ uptake into SR in cardiac homogenate with increasing free Ca2⫹ concentration was measured. Apparent affinity of SR Ca2⫹-pump for Ca2⫹ (EC50) and the maximum rate of Ca2⫹ uptake into SR (Vmax) are shown. Open bars, data from control rats (n ⫽ 3 hearts); closed bars, data from diabetic rats (n ⫽ 3 hearts). Data are means ⫾ SE of each group of rats. *P ⬍ 0.05 vs. control rats. AJP-Heart Circ Physiol • VOL 283 • OCTOBER 2002 • www.ajpheart.org DIABETIC CARDIOMYOPATHY ments (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 C2 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 AJP-Heart Circ Physiol • VOL H1407 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. 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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