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Journal of Physiology (2001), 537.1, pp. 83-92
© Copyright 2001 The Physiological Society
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ABSTRACT |
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INTRODUCTION |
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Diabetes mellitus is an increasingly prevalent chronic disease (Nathan et al. 1997) leading to several life-threatening complications. Cardiovascular disease often develops, and is the major cause of morbidity and mortality (Nathan et al. 1997; Wild et al. 1999). Diabetes-related death is associated with an increased susceptibility to cardiac arrhythmias (Ewing et al. 1991; Robillon et al. 1999), often reflected as changes in the electrocardiogram, both in insulin-dependent (type 1, IDDM) or in non-insulin-dependent (type 2, NIDDM) diabetes (Airaksinen, 1985; Lo et al. 1993). Decreased potassium currents in cardiac ventricular cells, with concomitantly prolonged action potentials, have been determined as underlying features of IDDM (Magyar et al. 1992; Shimoni et al. 1994). However, changes in electrophysiological characteristics have not been studied in detail in NIDDM, despite its considerably higher prevalence (Nathan et al. 1997; Wild et al. 1999). A prolongation of the action potential and an increased dispersion of the action potential repolarization, such as seen in IDDM, are major causes of cardiac arrhythmias (Surawicz, 1997; Robillon et al. 1999).
Diabetes also results in activation of a cardiac renin-angiotensin system (Sechi et al. 1994; Fiordaliso et al. 2000). Blockade of angiotensin II (ATII) receptors reverses some of the adverse effects of diabetes (Malhotra et al. 1997; Cao et al. 1998; Collis et al. 2000). We now report that the attenuation of ventricular K+ currents and the prolongation of the action potential in a rat model of IDDM can be reversed by blocking ATII receptors or by inhibiting the angiotensin-converting enzyme (ACE). Furthermore, we report for the first time that in the db/db mouse, a model of NIDDM (Chua et al. 1996), cardiac ventricular K+ currents are also attenuated. However, these currents can be significantly augmented by ATII receptor blockade. These results provide a rationale for the proven benefits of ACE inhibitors or ATII receptor blockers in diabetes (Zuanetti et al. 1997; Gerstein et al. 2000). In addition to ameliorating hypertension, these drugs may reduce the risk of cardiac arrhythmias.
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METHODS |
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All experiments were done in accordance with the guidelines of the Animal Care Committee of the University of Calgary.
Animal models
Two diabetic models were used in this work. Sprague-Dawley rats (220-280 g) were injected intravenously with streptozotocin (STZ, 100 mg kg-1) 6-13 days before experiments. Plasma glucose and insulin levels were determined and confirmed the diabetic state (hyperglycaemia and insulin deficiency). Age-matched uninjected rats were used as controls. In most experiments ventricular cells were isolated (as described in the following subsection) and then treated in vitro with various drugs. In one group, however, the drug valsartan was given in the drinking water (1 g l-1) for 5-10 days before STZ injection, and following STZ injection until the rats were killed. The NIDDM model used was the db/db mouse (Chua et al. 1996), a leptin receptor mutant that develops all the symptoms of (type 2) diabetes (hyperglycaemia, hyperinsulinaemia and insulin resistance). Mice were purchased from Jackson Labs (Mississauga, Ontario, Canada) and used at 12 weeks of age. Measurements of plasma glucose and insulin confirmed the diabetic state. Age-matched heterozygous db/+ mice were used as controls.
Cell isolation
Animals were anaesthetized with methoxyfurane and killed by cervical dislocation. Hearts were removed and the aortas cannulated for retrograde coronary perfusion (at 37 °C). The basic perfusing solution consisted of (mM): 121 NaCl; 5.4 KCl, 2.8 sodium acetate; 1 MgCl2; 5 Na2HPO4; 24 NaHCO3; and 5 glucose (bubbled continuously with a mixture of 95 % O2-5 % CO2). Initial perfusion was done with this solution including 1 mM CaCl2 (for 5 min). This was followed by 10 min perfusion with the basic solution with calcium omitted, followed by 7-8 min in the calcium-free solution containing collagenase (Yakult Honsha, Tokyo, Japan; 1.5 mg (100 ml)-1), protease (Sigma type XIV; 0.75 mg (100 ml)-1), taurine (20 mM) and 40 µM CaCl2. The free wall of the right ventricle was cut into small pieces for further incubation in a shaker bath at 37 °C, in a solution containing 0.3 mg ml-1 collagenase, 0.2 mg ml-1 protease, 20 mM taurine, 10 mg ml-1 albumin and 10 µM CaCl2 . Aliquots of cells were collected over 15-30 min, and divided into untreated or treated groups. Cells were stored (up to 9 h, at 20-22 °C) in the basic solution containing 20 mM taurine, 5 mg ml-1 albumin and 0.1 mM CaCl2.
Current and voltage recording
For each experiment, currents were recorded from treated and untreated cells. This allowed for small variations in the diabetic state. Current densities were obtained by dividing current magnitudes by cell capacitance. This was measured by integrating currents obtained in response to 5 mV steps from the holding potential (-80 mV). Current densities were averaged from all cells in each group, for each experiment. Cells were placed on the stage of an inverted microscope and superfused with a solution containing (mM): 150 NaCl, 5.4 KCl, 1 CaCl2; 1 MgCl2, 5.5 glucose; and 5 Hepes (pH 7.4). For current (but not action potential) measurements 0.3 mM CdCl2 was added, to block L-type calcium currents. The whole cell suction electrode method was used to record membrane currents (in the voltage-clamp mode) or action potentials (in the current-clamp mode), at 20-22 °C. Low resistance electrodes were used and series resistance was compensated for. Electrodes contained (mM): 120 potassium aspartate; 30 KCl, 4 Na2ATP; 10 EGTA, 1 CaCl2; 1 MgCl2; and 10 Hepes (pH 7.2). This solution creates a liquid junction potential of ~10 mV, which was corrected for. The presence of 0.3 mM Cd2+ in the perfusing solution, as well as EGTA in the pipette, ensured the recording of calcium-independent outward currents. Data was digitized (at 2-5 kHz), collected on-line and stored for subsequent analysis.
Drugs
STZ and saralasin were purchased from Sigma; valsartan and quinapril were a gift from Dr A. Gillis (Foothills Hospital and University of Calgary Cardiovascular Research Group).
Statistics
Comparisons between treated and untreated groups were made using one-way ANOVA, followed by a Student-Newman-Keuls multiple comparisons test. P values lower than 0.05 were taken to indicate significant differences.
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RESULTS |
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The first set of experiments was done using ventricular myocytes isolated from STZ-diabetic rats. The STZ model is commonly used to study type 1 diabetes (Tomlinson et al. 1992), which is characterized by hyperglycaemia and insulin deficiency. Two major repolarizing currents, a transient and a sustained K+ current, labelled It and ISS, respectively (and characterized by Apkon & Nerbonne, 1991), are attenuated in ventricular cells from these rats (Magyar et al. 1992; Shimoni et al. 1994, 1998). Some of this attenuation is due to the deficiency in insulin, since in vitro incubation with insulin for > 6 h can augment these currents, as shown in our earlier work (Shimoni et al. 1998). In the present work, we find that incubation of these cells with the non-specific (Burnier, 2001) angiotensin II receptor inhibitor saralasin (1 µM) for 6-9 h leads to a significant enhancement of both K+ currents, as shown in Fig. 1.
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Figure 1. The effects of ATII receptor blockade on outward currents in myocytes from STZ-diabetic rats A, superimposed current traces obtained in response to 500 ms depolarizing pulses given from a holding potential of -80 mV to potentials ranging from -20 to +50 mV. Examples are given for cells obtained from a control rat (left), and from an STZ-diabetic rat in the absence of (centre) or 7 h after incubation with 1 µM saralasin (right). B, current-voltage relationships for the peak outward current (It, left) and the sustained current (ISS, right) in cells from STZ-diabetic rats in the absence ( | ||
Sample current traces obtained from control and diabetic myocytes, the latter in the absence or presence of saralasin, are shown in Fig. 1A. Figure 1B shows the current-voltage relationships for the transient (peak outward) and sustained currents in diabetic myocytes, in the absence or presence of saralasin.
Subsequently, the more specific type 1 (Burnier, 2001) angiotensin receptor blocker valsartan was used. When isolated cells were incubated with 1 µM valsartan for 6-9 h the mean density of It and ISS was augmented. The mean densities of It (at +50 mV) in the absence and presence of valsartan were 18.9 ± 0.84 (n = 59) and 24.0 ± 0.98 pA pF-1 (n = 54), respectively (P < 0.0003). The values for ISS were 6.5 ± 0.25 and 8.1 ± 0.34 pA pF-1, respectively (P < 0.003). This result is shown in Fig. 2A.
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Figure 2. The in vitro and in vivo effects of the AT1 receptor blocker valsartan on outward currents in myocytes from STZ-diabetic rats A, mean (± S.E.M.) current densities (at +50 mV) for It (left) and ISS (right) obtained in myocytes from STZ-diabetic rats in the absence of (n = 59, open bars) or 6-9 h after incubation with 1 µM valsartan (hatched bars, n = 54). Asterisks denote significant differences (P < 0.0003). B, mean (± S.E.M.) current densities (at +50 mV) in control cells (open bars, n = 86), in cells from STZ-diabetic rats (hatched bars, n = 122) and in cells obtained from STZ-diabetic rats which had 1 g l-1 valsartan added to their drinking water 5-10 days prior and subsequent to STZ injection (cross-hatched bars, n = 97). Asterisks denote significant differences (* P < 0.01; **P < 0.001). Valsartan partially protected against the effects of STZ so that both It (left) and ISS (right) were significantly larger than in cells from STZ-treated rats without valsartan. However, both currents from diabetic valsartan-treated cells were still smaller (P < 0.001) than in control cells. | ||
In another group of rats, valsartan was given in vivo, administered for 5-10 days in the drinking water (1 g l-1), prior to induction of diabetes with STZ. The drug was maintained in the water until the rats were killed 6-10 days later. This short exposure to valsartan was sufficient to partly offset the effects of STZ, with a significant enhancement of both It and ISS. In 97 cells It density (at +50 mV) was 21.3 ± 0.68 pA pF-1, which is significantly (P < 0.0001) larger than in cells from STZ-diabetic rats without valsartan, in which the density was 17.5 ± 0.66 pA pF-1 (n = 122). However, the enhanced current was still significantly (P < 0.001) smaller than It in cells from control rats (25.0 ± 0.89 pA pF-1, n = 86). The corresponding values for ISS in myocytes from control, STZ-diabetic and diabetic valsartan-treated rats were 8.8 ± 0.25, 5.6 ± 0.14 and 6.3 ± 0.21 pA pF-1. The enhancement of ISS by valsartan was small but significant (P < 0.01). These results are shown in Fig. 2B.
These results are in accordance with earlier as well as more recent work suggesting that isolated cardiac myocytes release angiotensin II in an autocrine manner in response to stress (Zhang et al. 1995; Dostal, 2000; Barlucchi et al. 2001). ATII release was also shown to occur in myocytes obtained from (type 1) diabetic rats as well (Sechi et al. 1994; Fiordaliso et al. 2000). A pivotal finding made recently was that a prolonged exposure to ATII leads to an attenuation of a transient K+ current in canine cardiac myocytes (Yu et al. 2000). The present results therefore suggest that it is the release of ATII by ventricular cells in the STZ-diabetic rat that plays a major role in the attenuation of It and ISS, since these currents can be augmented if ATII receptors are blocked.
This hypothesis was further tested by experiments designed to establish whether an on-going formation of ATII by the angiotensin-converting enzyme (ACE) mediates the attenuation of K+ currents in isolated cells from diabetic rats. Experiments were done using quinapril (accupril), an ACE inhibitor. Incubation of ventricular cells, isolated from STZ-treated rats, with 1 µM quinapril for 6-9 h resulted in a highly significant (P < 0.0002) enhancement of the two K+ currents. This result is shown in Fig. 3A. In this group, the density of It (at +50 mV) was 18.6 ± 1.03 (n = 52) and 24.8 ± 1.25 pA pF-1 (n = 45) in the absence and presence of quinapril, respectively. The corresponding values for ISS were 6.7 ± 0.27 and 8.9 ± 0.47 pA pF-1. The enhancement of outward currents by quinapril also led to an enhanced repolarization of the action potential. Thus, the prolongation of the ventricular action potential in STZ-diabetic rats (due to the attenuated outward currents) could be reversed by quinapril. Figure 3B shows action potentials in STZ-diabetic rats, in the absence or following 7 h in quinapril. The ACE inhibitor clearly abbreviates the action potential. The summary data for action potential duration (at -60 mV) is shown in panel C.
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Figure 3. The effects of inhibiting the angiotensin-converting enzyme (ACE) on outward K+ currents and action potentials in cells from STZ-diabetic rats A, mean (± S.E.M.) current densities (at +50 mV) of It (left) and ISS (right) in the absence of (open bars, n = 52) or following incubation (6-9 h) with 1 µM quinapril (hatched bars, n = 45). B, action potentials obtained in single ventricular cells (at a 1 Hz stimulation rate) from an STZ-diabetic rat, either with no drug added (left) or after 7 h in 1 µM quinapril (right). C, mean (± S.E.M.) values for action potential duration (at -60 mV) in cells from STZ-diabetic rats in the absence of (open bars, n = 28) or after incubation (6-9 h) with 1 µM quinapril (hatched bars, n = 17). Asterisks denote significant differences (* P < 0.03; **P < 0.0002). | ||
The results described so far indicate that the inhibition of either angiotensin II action or its formation augments the attenuated outward currents in IDDM, with a concomitant abbreviation of the ventricular action potential. Since these results were obtained by measuring currents after > 6 h incubation, it was important to establish whether there are acute effects on It and ISS. Figure 4 shows that exposure of a myocyte obtained from an STZ-diabetic rat to 1 µM quinapril for up to 25 min has no effects on either It or ISS, or on the background inwardly rectifying current IK1. The same result was seen in six additional cells. Thus, the effects described previously are obtained only after a prolonged exposure to the drug.
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Figure 4. Absence of an acute effect of quinapril on outward currents A, superimposed currents obtained in response to pulses from -80 to +50 mV (upward deflections) or to -110 mV (downward traces) before and 25 min after addition of 1 µM quinapril to the perfusing solution. B, current amplitudes plotted against time, showing no changes in It, ISS or IK1 following the addition of 1 µM quinapril. | ||
The following experiments addressed several additional issues. In the experiments described previously recordings were made after exposing cells to drugs for up to 9 h. Earlier work by Mitcheson et al. (1996) showed that cell capacitance decreases within 24 h following isolation. A comparison of cell capacitance in the first 1-5 h after isolation and 6-9 h after isolation (in the presence of valsartan or quinapril) showed that there were no changes in cell capacitance over this time period. Thus, in the first 1-5 h cell capacitance in cells from STZ-diabetic rats was 57.5 ± 1.54 pF (n = 104), whereas in cells treated with 1 µM valsartan or 1 µM quinapril for 6-9 h, the capacitance was 55.7 ± 1.69 (n = 97) and 56.6 ± 1.30 pF (n = 99), respectively.
In addition, the possibility that the changes observed in current magnitude were due to changes in the gating characteristics of these K+ channels was considered. Yu et al. (2000) found that prolonged exposure to angiotensin II changes the recovery of It from inactivation, as well as the steady-state inactivation. The steady-state inactivation of It was measured using a standard double-pulse protocol in cells from STZ-diabetic rats before and 6-9 h after exposure to 1 µM quinapril. Figure 5 shows the results, obtained in the absence (n = 23 cells) or presence (n = 22) of quinapril. Panel A shows the voltage protocol, in which 500 ms prepulses were given to potentials ranging from -100 to +20 mV, followed by a pulse to +50 mV. Prepulse increments of 10 mV were used, except in the steeper part of the inactivation process, where 5 mV steps were used. Panels B and C show the currents in response to these steps in the absence or presence of quinapril (in which case the currents are enhanced). It in these experiments was measured as the difference between peak and steady-state current, and current magnitude was normalized as a fraction of the maximal current. Curve fitting (assuming a Boltzmann distribution) yielded V0.5 (membrane potential for 50 % activation) and slope values of, respectively, -33.6 ± 0.4 mV and 5.99 ± 0.4 in the absence of quinapril and -34.3 ± 0.5 mV and 6.64 ± 0.4 in its presence. Panel D shows the summary data (mean ± S.E.M.), with the lines drawn using parameters obtained by best curve fitting.
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Figure 5. Steady-state inactivation of It in cells from STZ-diabetic rats is unaffected after 6-9 h in 1 µM quinapril A, voltage protocol used for measuring inactivation: 500 ms prepulses to potentials ranging from -100 to +20 mV preceded a 500 ms test pulse to +50 mV. The currents elicited by test pulses gradually decline. B, current traces in the absence of quinapril. C, currents in a cell exposed for 7 h to quinapril. D, currents were normalized as fractions of the maximal current elicited. Mean (± S.E.M.) ratios are plotted against membrane potential. Curves obtained by fitting are superimposed (parameters in text), showing no effects of quinapril on steady-state inactivation of It. | ||
Recovery from inactivation was measured by applying double pulses (to +50 mV), separated by varying intervals, ranging from 10 to 800 ms. Figure 6 shows the recovery of It in the absence of quinapril (panel A) or after 6 h in quinapril (panel B). The currents (peak steady state) were normalized as ratios of the magnitude of the current in response to the second pulse, divided by the magnitude of the current obtained in response to the first pulse. The data were best fitted by a mono-exponential curve, with time constants of 78.3 ± 4.3 and 79.5 ± 4.3 ms in the absence and presence of quinapril, respectively. The mean recovery values and the plots using the time constants obtained by fitting the data to a mono-exponential process are shown in panel C. This clearly demonstrates an absence of any effect of quinapril on the recovery process.
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Figure 6. The recovery of It from inactivation in cells from STZ-diabetic rats is unchanged by quinapril Superimposed current traces obtained in response to double pulses (to +50 mV) given at intervals ranging from 10 to 800 ms are shown. A, recovery in the absence of quinapril. B, It recovery in a cell exposed for 6 h to 1 µM quinapril. C, mean (± S.E.M.) normalized relative current values (indicating recovery) plotted against interpulse intervals. Superimposed are the plots obtained by fitting the points to a mono-exponential process (parameters are given in the text). | ||
The lack of an acute effect of quinapril on current magnitude, as well as the absence of changes in gating parameters, suggested that the augmentation of currents is dependent on the synthesis of new K+ channels. An indirect way of testing this was to ascertain if the effects of quinapril and valsartan depend on protein synthesis, as was found to be the case in our earlier work with insulin (Shimoni et al. 1998).
In subsequent experiments 2 µM cycloheximide was added to cells 1 h before the addition of 1 µM quinapril. Currents were measured 6-9 h after addition of quinapril. The enhancement of It and ISS was significantly reduced (P < 0.05 and P < 0.001, respectively). The mean densities (at +50 mV) of It were 16.3 ± 0.8 (n = 74), 20.1 ± 0.8 (n = 62) and 17.6 ± 0.9 pA pF-1 (n = 57) in the absence of quinapril, with quinapril, and with quinapril and cycloheximide, respectively. The corresponding values for ISS were 5.2 ± 0.2, 6.8 ± 0.2 and 5.7 ± 0.2 pA pF-1. These results are shown in Fig. 7A. Similar results were obtained when valsartan was added after exposure of cells to cycloheximide, as shown in Fig. 7B.
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Figure 7. The effects of quinapril and valsartan are blocked by cycloheximide A, mean current densities (at +50 mV) are shown (It on the left, ISS on the right) in the absence of quinapril (open bars, n = 74), following 6-9 h in 1 µM quinapril (hatched bars, n = 62), and with quinapril and 2 µM cycloheximide added 1 h before quinapril (cross-hatched bars, n = 57). Cycloheximide significantly reduced the effects of quinapril on both It (P < 0.05) and ISS (P < 0.001), making these currents not significantly different from those in untreated cells. B, mean densities in the absence of valsartan (open bars, n = 59), after 6-9 h in 1 µM valsartan (hatched bars, n = 56), and with valsartan and cycloheximide (cross-hatched bars, n = 44). In this set of experiments, cycloheximide also significantly reduced the quinapril-produced augmentation of It (P < 0.05) and ISS (P < 0.01), but ISS was still larger than in untreated cells (P < 0.001). It in valsartan and cycloheximide was not different from that in untreated cells. * P < 0.05; ** P < 0.01. | ||
It was of major interest to establish whether similar mechanisms are operative in NIDDM, since the vast majority of diabetic patients (80-90 %) have this type of diabetes (Nathan et al. 1997; Wild et al. 1999). NIDDM is characterized by peripheral insulin resistance and hyperinsulinaemia, as well as by secretory defects in pancreatic 
-cells. Although some ECG abnormalities and associated arrhythmias are similar to those measured in type 1 diabetes (Robillon et al. 1999), the underlying ion current changes in cardiac cells have not been determined, to our knowledge. In the present work we used the db/db mouse, a leptin receptor mutant that, when homozygous, develops obesity and all the symptoms of NIDDM (Chua et al. 1996). These mice are extremely hyperinsulinaemic, in contrast to the hypoinsulinaemic STZ-diabetic rats. Despite the differences in insulin levels, isolated myocytes from db/db mice (at 12 weeks of age) exhibit attenuated peak and sustained outward currents, as compared to age-matched lean db/+ mice. This result is shown in Fig. 8A. Current traces from a myocyte from a db/db mouse are shown (right) along with traces from a myocyte obtained from a lean heterozygous db/+ mouse (left). The mean ± S.E.M. densities (at +50 mV) are shown in panel B, with the peak current on the left and the sustained current on the right. Concordant with the reduction in outward currents, the action potentials in myocytes from the db/db mouse are prolonged. Sample action potentials are shown in panel C, obtained in cells from a db/+ (left) or db/db (centre) mouse. The summary data for action potential duration at -60 mV is shown on the right of panel C.
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Figure 8. Outward currents and action potentials in the db/db mouse A, superimposed current traces obtained in response to 500 ms pulses given from -80 mV to potentials ranging from -10 to +50 mV in ventricular myocytes obtained from a lean heterozygous db/+ mouse (left) or from an age-matched obese db/db mouse (right). B, current densities (mean ± S.E.M.) for peak outward current (left) and sustained current (right), measured at the beginning and end of pulses to +50 mV, respectively. Open bars, cells from db/+ mice (n = 24); hatched bars, cells from db/db mice (n = 35). C, samples of action potentials obtained at a stimulation rate of 1 Hz from myocytes obtained from a db/+ mouse (left) or db/db mouse (centre). The panel on the right shows the mean (± S.E.M.) action potential duration (at -60 mV) obtained in cells from the two groups (open bars; db/+, n = 13; hatched bars; db/db, n = 17). ** P < 0.002. | ||
Thus, despite the drastically different levels of circulating insulin levels in the STZ-diabetic rat and in the db/db mouse (reduced and augmented relative to normal, respectively), the changes in outward K+ currents and the ventricular action potential are very similar. We examined whether an enhanced action of ATII is possibly an underlying mechanism for the changes in K+ currents in the db/db mouse, as is presumably the case in the STZ-diabetic rat. Incubation of isolated myocytes from the db/db mouse with the ATII receptor blocker valsartan (1 µM, > 6 h) produced a significant enhancement of both the peak (P < 0.0003) and the sustained (P < 0.004) outward currents, as shown in Fig. 9. Panel A shows superimposed current traces, given to -110 mV and to potentials ranging from -10 to +50 mV, in two myocytes from a db/db mouse, in the absence (left) or presence (right) of valsartan (1 µM, 7 h). The summary data for current density at +50 mV is shown in panel B (peak current on left, sustained current on right).
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Figure 9. The effects of valsartan on outward currents in myocytes from the db/db mouse A, superimposed current traces, obtained in response to depolarizing pulses from -80 mV to potentials ranging from -10 to +50 mV, as well as in response to a pulse to -110 mV, are shown in myocytes from a db/db mouse in the absence of (left) or following 7 h in 1 µM valsartan (right). B, mean (± S.E.M.) current densities for peak (left) and sustained (right) currents obtained from cells in the absence (open bars, n = 27) or presence (hatched bars, n = 21) of 1 µM valsartan (6-9 h incubation). Asterisks denote significant differences (P < 0.003). | ||
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DISCUSSION |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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In combination, these results provide further confirmation of earlier suggestions and direct measurements showing that in both IDDM and NIDDM there is an activation of the cardiac renin-angiotensin system (Sechi et al. 1994; Collis et al. 2000; Fiordaliso et al. 2000). The novelty of the present findings is in the functional consequences of this activation. The enhanced, chronic autocrine release of ATII by ventricular cells leads to the attenuation of two major repolarizing potassium currents in cardiac ventricular cells. This, in turn, prolongs the action potential and is presumably at least partly responsible for the prolongation of the QT interval in the diabetic electrocardiogram. If either the formation or the action of ATII is blocked, an inhibitory effect exerted by ATII is removed, enabling the recovery of It and ISS magnitudes towards their normal, non-diabetic values. This is the first direct demonstration that pathological changes in ion currents can be reversed by reducing the action of angiotensin II, either by blocking its receptors or by reducing its formation through the angiotensin-converting enzyme pathway. The mechanism by which this occurs is not known at present and needs to be further investigated. The excess ATII may prevent synthesis, assembly or post-translational modification of the K+ channel proteins, as well as the correct targeting to the cell membrane. The inhibition of current enhancement by both valsartan and quinapril when cycloheximide is present suggests that excess ATII is associated with the inhibition of protein synthesis, possibly of the K+ channels themselves. Furthermore, it is now well established that accessory subunits such as KCHIP2 play a major role in regulating or modifying the expression or function of the transient outward current (Patel et al. 2001; Rosati et al. 2001). Thus, it is possible that excess ATII modifies the action of this or other accessory subunits. The blocking of ATII action or formation may restore the normal interaction between all the regulatory factors that produce the normal current phenotype. These issues must be addressed directly in future experiments.
One probable mediator of ATII action is the second messenger protein kinase C (PKC) (Dostal, 2000). Acute activation of the 
isoform of PKC has been shown to attenuate a transient and sustained K+ current It and ISS (Shimoni, 1999), but this effect is absent in STZ-diabetic myocytes (Shimoni, 1999). The subcellular distribution and activity of PKC is altered in both IDDM and NIDDM (Avignon et al. 1996; Malhotra et al. 1997), with an impaired PKC translocation found in patients with NIDDM (Nagy et al. 1991). These effects may be partly due to activation of the renin-angiotensin system, since ATII receptor blockade has been shown to restore the normal subcellular distribution of PKC (Malhotra et al. 1997), as well as reversing some of the derangements associated with diabetes (Collis et al. 2000). However, the changes occurring at the subcellular level may have more subtle complexity. Thus, Buttrick (1998) has recently reported that prolonged exposure to ATII alters the expression of a PKC anchoring protein, with no change in membrane or cytosolic PKC per se.
The present results show for the first time that both type 1 and type 2 diabetes lead to a comparable attenuation in membrane potassium currents along with a prolongation of the ventricular action potential. This attenuation is also reversed by ATII receptor blockade, suggesting that activation of the ATII pathway may be a common feature to metabolic stress. It is not known at present whether other pathways of angiotensin II formation, such as described recently by Barlucchi et al. (2001), are involved. Beneficial effects on cardiac electrical activity due to ATII blockade have been suggested to occur in a model of heart failure (Spinale et al. 1997). Indirect evidence for an increase in calcium channel density (assessed by binding experiments), as well as a restoration of action potential duration to normal values was observed following ATII receptor blockade or ACE inhibition (Spinale et al. 1997, 1998). Thus, common pathways may be activated in several pathophysiological conditions, often leading to changes in action potential configuration. Changes in action potential repolarization are a well-established mediator of lethal cardiac arrhythmias (Ewing et al. 1991; Surawicz, 1997; Robillon et al. 1999). These results therefore suggest that the established benefits of ATII blockers or ACE inhibitors in diabetes may be largely due to the effects described here, since the augmented currents and shorter action potentials would reduce the susceptibility to ventricular arrhythmias.
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REFERENCES |
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| AIRAKSINEN, K. E. J. (1985). Electrocardiogram of young diabetic patients. Annals of Clinical Research 17, 135-138 | [Medline] |
| APKON, M. & NERBONNE, J. M. (1991). Characterization of two distinct depolarization-activated K+ currents in isolated adult rat ventricular myocytes. Journal of General Physiology 97, 973-1011 | [Abstract] |
| AVIGNON, A., YAMADA, K., ZHOU, X., SPENCER, B., CARDONA, O., SABA-SIDDIQUE, S., GALLOWAY, L., STANDAERT, M. L. & FARESE, R. V. (1996). Chronic activation of protein kinase C in soleus muscles and other tissues of insulin-resistant type II diabetic Goto-Kakizaki (GK), obese/aged and obese/Zucker rats. Diabetes 45, 1396-1404 | [Abstract] |
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Acknowledgements
This work was supported by a grant from the Canadian Diabetes Association in honour of Mary Selina Jamieson, and by the Heart and Stroke Foundation of Alberta. I would like to thank Dr J. Guo for his help in cell isolation.
Correspondence
Y. Shimoni: Department of Physiology and Biophysics, Health Sciences Centre, University of Calgary, 3330 Hospital Drive NW, Calgary, Alberta, Canada T2N 4N1.
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, n = 24) and presence (
, n = 23) of saralasin (6-9 h incubation). Mean current density values (± S.E.M.) are plotted against membrane potential.