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Am J Physiol Heart Circ Physiol 281: H1800-H1807, 2001;
0363-6135/01 $5.00
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Vol. 281, Issue 4, H1800-H1807, October 2001

Altered K+ channel gene expression in diabetic rat ventricle: isoform switching between Kv4.2 and Kv1.4

Atsushi Nishiyama1, Douglas N. Ishii1,2, Peter H. Backx3, Bruce E. Pulford1, Barbara R. Birks1, and Michael M. Tamkun1,2

1 Departments of Physiology and 2 Biochemistry and Molecular Biology, Colorado State University, Ft. Collins, Colorado, 80523; and 3 Department of Medicine, Centre for Cardiovascular Research, and the Toronto Hospital, University of Toronto, Toronto, Canada M5G 2C4


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Expression of voltage-gated K+ channels encoding the K+ independent transient outward current in the streptozocin-induced diabetic (DM) rat ventricle was studied to determine the basis for slowed cardiac repolarization in diabetes mellitus. Although hypertrophy was not detected in diabetic rats at 12 wk after streptozocin treatment, ventricular Kv4.2 mRNA levels decreased 41% relative to nondiabetic controls. Kv1.4 mRNA levels increased 179% relative to controls, whereas Kv4.3 mRNA levels were unaffected. Immunohistochemistry and Western blot analysis of the diabetic heart showed that the density of the Kv4.2 protein decreased, whereas Kv1.4 protein increased. Thus isoform switching from Kv4.2 to Kv1.4 is most likely the mechanism underlying the slower kinetics of transient outward K+ current observed in the diabetic ventricle. Brain Kv1.4, Kv4.2, or Kv4.3 mRNA levels were unaffected by diabetes. Myosin heavy chain (MHC) gene expression was altered with a 32% decrease in alpha -MHC mRNA and a 259% increase in beta -MHC mRNA levels in diabetic ventricle. Low-dose insulin-like growth factor-II (IGF-II) treatment during the last 6 of the 12 wk of diabetes (DM + IGF) protected against these changes in MHC mRNAs despite continued hyperglycemia and body weight loss. IGF-II treatment did not change K+ channel mRNA levels in DM or control rat ventricles. Thus IGF treatment may prevent some, but not all, biochemical abnormalities in the diabetic heart.

potassium channel expression; diabetes; insulin-like growth factor; hypertrophy


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE DIABETIC HEART is attended by increased mortality. The cardiovascular complications are well known in both human and experimental diabetes mellitus (16, 25, 43, 54). There are distinct alterations in both the mechanical and electrophysiological properties of the myocardium (13, 14, 32). The purpose of this study was to examine the biochemical pathology that may contribute to the mechanical and electrophysiological dysfunction in the diabetic heart.

Cardiac contractility (expressed as ventricular pressure development and peak pressure) is reduced, and relaxation time and velocity are markedly prolonged in experimental diabetes (15, 44, 45). In concordance with these mechanical changes, changes in electrocardiogram and action potential configuration are often observed in diabetic patients (20, 37). The most prominent electrophysiological alteration is the prolongation of the Q-T interval due to an increase in ventricular action potential duration (APD) (24, 38, 49). Recent electrophysiological findings have shown that the density of the K+ independent transient outward current (ITO) is reduced and the recovery kinetics of ITO slowed in diabetic myocardium relative to normal tissue. These changes in ITO properties are likely to be responsible for the APD prolongation in diabetic rats (48, 50). However, the molecular mechanisms responsible for these changes in ITO have not been examined.

Cardiac Kv1.4 and Kv4.2 channels were first proposed to be responsible for the ITO based solely on functional parameters (3, 41). Recently, Kv1.4, Kv4.2, and Kv4.3 were confirmed as responsible for the native ITO by gene knockout, antisense, and dominant negative approaches (19, 62). The primary difference between Kv1.4 and the two Kv4 channels is in the recovery from inactivation, with Kv1.4 recovering much more slowly than Kv4.2. Changes in outward current observed in diabetic animals could result from a decrease in the expression of Kv4.2 and an increase in the more slowly recovering Kv1.4 isoform. One aim of this study was to test the hypothesis that Kv4.2 expression is reduced in diabetes mellitus, along with a compensatory increase in Kv1.4 expression.

The second aim was to determine whether insulin-like growth factor (IGF) treatment can prevent or reverse the biochemical pathology associated with the diabetic ventricle. Circulating and tissue IGF levels are reduced in clinical (52, 59) as well as experimental (4, 11, 39) diabetes. IGF gene expression is reduced in diabetic nerves (60), and IGF administration can protect against diabetic neuropathy (21, 22, 64). Because IGF mRNA is reduced in cardiac muscle in diabetes (28), these observations together prompted us to determine whether IGF treatment could be cardioprotective. The expression of myosin heavy chain (MHC) isoforms is altered in diabetic cardiac muscle (35). Because altered isoform expression may contribute to mechanical dysfunction, the hypothesis that IGF treatment can prevent abnormal expression of MHC genes as well as Kv channel genes was tested.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Materials. The affinity-purified Kv1.4 antibody, affinity-purified Kv4.2 antibody, and Kv4.2-specific antiserum have been previously characterized (6, 58). Immunological reagents were purchased from Jackson Immunoresearch Laboratories.

Induction of diabetes and tissue preparation. All studies involving rats were done in accordance with the principles set forth in the Guide for the Care and Use of Laboratory Animals (National Institutes of Health, 1996). Male Sprague-Dawley rats (12 wk old) were randomly assigned to nondiabetic (control), diabetic (DM), and DM + IGF-II treatment (DM + IGF) groups. Each group contained four rats. After the rats were fasted overnight, diabetes was induced in rats with a single intraperitoneal injection of 50 mg/kg streptozotocin (STZ; Sigma) and maintained for 12 wk with free access to rat chow and water. At the end of 6 wk, the STZ-treated animals were implanted subcutaneously in the midback with osmotic minipumps (0.5 µl/h) that released either vehicle (1 mM acetate, pH 6) or IGF-II (5 µg · rat-1 · day-1) for the remaining 6 wk. Serum glucose concentrations were determined at the end of the first 6-wk period and on the day of euthanasia with the use of a glucose diagnostic kit (model 510A; Sigma). The cardiac ventricle and brain in each animal were removed, and small pieces of the left ventricular free wall near the left descending artery were embedded in Tissue Tek (Baxter) and slowly frozen at -80°C for immunohistochemical analysis. The remaining ventricular tissue was used for both membrane preparation and RNA extraction. One brain hemisphere (except for the olfactory bulb) was used for RNA extraction.

Northern blot analysis. Total RNA was extracted from freshly isolated ventricles and brains using the guanidinium thiocyanate method (1). Ethidium bromide (40 µg/ml) was added to RNA samples before electrophoresis to enable visual confirmation of RNA integrity. After fractionation of 10 µg of total RNA through a 1% agarose-3% formaldehyde gel in 20 mM MOPS and 1 mM EDTA, pH 7.4, the RNA was transferred to a Nytran filter (Schleicher and Schuell) by capillary action in 20× saline sodium citrate. RNA was cross-linked to the filter by ultraviolet irradiation using a Stratalinker (Stratagene). The filter was hybridized with a rat alpha - or beta -MHC oligonucleotide probe (Calbiochem). Oligonucleotide probes were 5' end labeled with [gamma -32P]ATP (6,000 Ci/mmol; ICN) using T4 polynucleotide kinase. The hybridization and wash were performed according to standard protocols. After hybridization and wash, the filter was exposed to a PhosphorImager screen (Molecular Dynamics) for quantification and then were exposed to XAR-5 film (Kodak).

Ribonuclease protection assay. The sequence of the cDNA templates used for the production of the radioactive antisense RNA probes for Kv1.4, Kv4.2, and Kv4.3 and the detailed procedure of ribonuclease protection assay (RPA) have been described previously (36). In brief, plasmids containing Kv channel cDNAs were linearized with the appropriate restriction enzyme and antisense cRNA probes were synthesized using the MAXIscript kit (Ambion) with [alpha -32P]UTP (ICN Radiochemicals). The cyclophilin cRNA probe was synthesized from cDNA (pTRI-cyclophilin rat antisense control template) purchased from Ambion to detect cyclophilin mRNA as an internal control. RPA was performed using a RPAIII kit (Ambion) according to the manufacturer's protocol. Hybridization of the Kv channel and cyclophilin probes with 10 µg total sample RNA was carried out at 42°C overnight, followed by digestion with a RNase A and T1 mix at 37°C for 30 min. The reaction was terminated by the addition of SDS and proteinase K. After ethanol precipitation, the protected RNA fragments were subjected to electrophoresis in a 5% polyacrylamide-8 M urea gel. The gel was dried on 3M paper and subjected to quantitative analysis using a PhosphorImager. The mRNA levels were normalized to the levels of cyclophilin mRNA and expressed in relative arbitrary units.

Immunofluorescence. Cryosections of the rat ventricle (10 µm) were incubated with primary rabbit antibody followed by biotin-conjugated goat anti-rabbit IgG (Jackson Immunoresearch) and CY3-conjugated streptavidin as previously described in detail (34). Samples were analyzed using a Nikon E800 microscope equipped with standard epifluorescence and a Princeton Instruments charge-coupled device camera. Antigen-blocking experiments were conducted to confirm Kv4.2 antiserum specificity. Here, the antibody staining was performed as described above except that cryosections were stained with antiserum that had been preincubated for 2 h at room temperature with 200 nmol of the Kv4.2 peptide per milliliter of diluted antiserum (34). Blocking experiments were not necessary with the other two antibodies because they were affinity purified.

Western blot analysis. Approximately 0.5 g of each thawed rat ventricle was homogenized with a high-shear homogenizer (Tekmar) in 5 ml of 0.32 M sucrose-5 mM Na2HPO4 with the following protease inhibitors (Sigma, unless otherwise noted): 0.31 mg/ml benzamidine, 0.62 mg/ml N-ethylmaleamide, 1 mg/ml bacitracin, 1 µg/ml pepstatin, 1 µg/ml leupeptin, 0.31 mg/ml pefabloc (Boehringer Mannheim), and 0.8 µg/ml calpain inhibitor (Calbiochem). Tissue was homogenized on ice at high speed with a small (1.0 cm) diameter probe for 60 s. The homogenate was centrifuged at 4°C for 10 min at 3,000 rpm (Beckman JA 25.5 rotor) to remove large debris and nuclei. The supernatant was further homogenized by 10 strokes in a 7-ml Wheaton dounce tissue grinder on ice and centrifuged for 1 h at 4°C and 12,000 rpm (JA 25.5; Beckman Instruments). The resulting membrane pellet was resuspended in 100 µl of ice-cold PBS and stored at -80°C. Membrane proteins were fractionated by SDS-PAGE and transferred overnight to nitrocellulose membrane (Schleicher & Schuell) using the standard Laemmli method as previously described (33). Briefly, SDS sample buffer was added to the isolated membranes before electrophoretic separation on a 10% polyacrylamide gel using a minigel system (Bio-Rad). After overnight transfer onto nitrocellulose membrane, the samples were stained with 0.1% Ponceau S Solution (Sigma) (to visualize the quality of the transfer), rinsed with deionized water, and incubated overnight at 4°C in solution 1 (S1) [50 mM Tris (pH 7.5), 150 mM NaCl, and 0.1% Tween-20, and 5% nonfat dry milk] to block nonantigenic sites. The blots were then incubated in S1 at room temperature for 1 h with either Kv1.4 or Kv4.2 polyclonal antibodies (diluted 1:1,000). The blots were washed 3 times for 15 min each with solution 3 (S3) [50 mM Tris (pH 7.5), 500 mM NaCl, and 0.1% Tween 20] and then incubated for 1 h at room temperature in S1 containing a 1:3,000 dilution of horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG or 1:5,000 of HRP-conjugated goat anti-mouse IgG. The blots were washed three times for 15 min each with S3 and detection was achieved with the Renaissance enhanced chemiluminescence reagent (New England Nuclear Life Science Products) per the manufacturer's instructions.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Body weights, ventricular weights, and serum glucose levels in diabetic animals. A single STZ treatment was used to experimentally induce the diabetic state over a 12-wk period. As summarized in Table 1, rats receiving STZ had significantly elevated serum glucose concentrations and reduced body weights compared with nondiabetic control animals. Low-dose IGF treatment (DM + IGF) affected neither serum glucose levels nor weight loss in the diabetic animals. There was no significant difference in ventricle weights among the experimental groups. The ventricle-to-body weight ratio increased in both DM groups relative to control because body weight was reduced by the diabetic condition, i.e., it was not indicative of cardiac hypertrophy. We also examined the ventricle and myocyte morphology (data not shown). There were no significant changes in ventricular wall thickness (control, DM, and DM ± IGF: 2.23 ± 0.14, 2.12 ± 0.16, and 2.26 ± 0.28 mm, respectively) or myocyte width (control, DM, and DM + IGF: 17.7 ± 2.2, 18.5 ± 2.7, and 18.3 ± 3.6 µm, respectively) among the groups (data not shown) (n = 40 myocytes total per group, each group consisting of four animals). These data indicate that diabetes, with or without IGF treatment, was not associated with cardiac hypertrophy using standard morphological measurements.

                              
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  		Table 1.   Effect of diabetes and IGF-II treatment on body wt, ventricle wt, and serum glucose levels

MHC isoform switching from the alpha - to beta -isoform occurs in the hypertrophied or diseased rat heart (23, 46). Therefore, although gross ventricular hypertrophy was not detected, we tested for altered myosin gene expression. The ventricular alpha - and beta -MHC mRNA levels as a function of treatment are shown by the Northern blot analysis of Fig. 1, A and B. Diabetic treatment resulted in a significant decrease of alpha -MHC mRNA to 32 ± 11% of control (P < 0.01), and IGF treatment restored the mRNA level to 51 ± 3% (P < 0.01 vs. DM) (Fig. 1A). Diabetic treatment increased the beta -MHC mRNA level to 259 ± 8% of control (P < 0.01), and IGF treatment reduced this change to 217 ± 31% of control (P < 0.05 vs. DM) (Fig. 1B). Thus hypertrophy may have developed with a longer time period, for myosin isoform expression did change.


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  		Fig. 1.   Effect of diabetes and insulin-like growth factor (IGF) treatment on myosin heavy chain (MHC) isoform mRNA levels in ventricle. The expression of cardiac alpha -MHC and beta -MHC mRNA in rat ventricle was determined by Northern blot analysis as shown in A and B, top, respectively. Shown are representative autoradiographs from two rats from each experimental group. MHC isoform mRNAs were normalized to 28S ribosomal mRNA levels and expressed as arbitrary units (%control) in A and B, bottom. C, control; DM, diabetic. Values are means ± SD (n = 4 rats per group). *P < 0.05; **P < 0.01.

Effect of diabetes and IGF treatment on potassium channel alpha -subunit mRNA levels in heart and brain. To examine the effect of diabetes and IGF treatment on Kv1.4, Kv4.2, and Kv4.3 gene expression in the heart, mRNA levels were measured in ventricles using an RNase protection assay with cyclophilin mRNA used as an internal control. As shown in Fig. 2, A and B, Kv1.4 mRNA level increased to 179 ± 24% of control (P < 0.01), whereas Kv4.2 mRNA level decreased to 41 ± 5% of control (P < 0.01) in the diabetic rat ventricles. IGF treatment did not reverse these changes. In contrast, Kv4.3 mRNA levels showed no significant change with any treatment (Fig. 2C).


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  		Fig. 2.   Change in K+ channel mRNA levels in diabetic ventricles. Kv1.4, Kv4.2, and Kv4.3 mRNA levels were measured in control, DM, and (DM + IGF) rat ventricles as shown in A, B, and C, respectively. Ventricular total RNA (10 µg) was subjected to RNase protection assay using 32P-labeled Kv and cyclophilin (cyclo) cRNAs as probes as described in MATERIALS AND METHODS. A-C, left: representative autoradiographs obtained with each experimental group. Open arrowheads indicate the position of undigested Kv and cyclophilin (cyclo) cRNA probes, and closed arrowheads indicate the protected fragments. Kv mRNA was normalized to cyclophilin mRNA levels and expressed as relative arbitrary units (A-C, right). Presented are means ± SD (n = 4 rats per group). **P < 0.01.

To determine whether change in K+ channel mRNA levels in diabetes was restricted to the heart, mRNA levels of Kv1.4, Kv4.2, and Kv4.3 were examined also in the brain. As shown in Fig. 3, A-C, diabetic animals had the same relative mRNA levels for all three channels in the brain. Two alternatively spliced variants of Kv4.3 are present in the rat brain, whereas only a single isoform is found in the ventricle (36, 51). Diabetes had no effect in relative levels of these two splice variants in the brain.


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  		Fig. 3.   K+ channel mRNA levels in diabetic brain. Kv1.4 (A) and Kv4.2 (B) mRNA levels were measured by RNase protection in C, DM, and (DM + IGF) rat whole brains. C: expression of Kv4.3 splice variants. Left: autoradiographs; right: Kv mRNA expressed as relative arbitrary units. With the RNase protection assay probe used in this study, the protected fragment for Kv4.3 long shows 312 bp and that for Kv4.3 short shows 165 and 90 bp (36). The 90-bp band is hidden by the robust cyclophilin mRNA band (103 bp). Presented are means ± SD (n = 4 rats per group).

Immunoreactive Kv4.2 and Kv1.4 protein levels in diabetic ventricle. Kv alpha -subunit protein expression levels do not always correlate with mRNA levels in rat heart (61). In addition, altered expression could be occurring in cell types other than ventricular myocytes. Therefore, Kv4.2 immunostaining of ventricle cryosections was performed to address the location and amount of ventricular K+ channel protein levels in the diabetic state. In the control rat ventricle, Kv4.2 immunoreactivity was distributed evenly over myocytes, while being slightly concentrated at the ends of the cells [Fig. 4A, consistent with previous reports (6)]. In the diabetic rat ventricle, the staining level was reduced compared with control (compare Fig. 4, A and B) as predicted from the changes in mRNA levels. The antibody staining of IGF- and vehicle-treated diabetic rat ventricles was similar (data not shown), again as predicted from the mRNA analysis. Figure 4, C and D, shows the staining observed with no primary antibody or with antiserum previously adsorbed with the peptide used for antiserum production. Only background staining was observed in the absence of primary antibody, and the peptide treatment reduced the staining to near-background levels. Together these data indicate antibody specificity. Also supporting the antibody specificity are the results shown in Fig. 4, E and F. Here, a different and affinity-purified anti-Kv4.2 antibody was used to stain control and diabetic myocardium, Fig. 4, E and F, respectively. Again, antibody staining was reduced in the diabetic tissue. Figure 4, G and H, illustrates the immunostaining observed with an affinity-purified anti-Kv1.4 antibody. As predicted from the changes in mRNA levels, the diabetic state caused an increase in Kv1.4 protein as detected by the immunofluorescence. Figure 4G shows staining to control tissue, whereas Fig. 4H represents diabetic tissue. Although gradients of Kv1.4 and Kv4.2 expression exist across the ventricular wall (6, 8, 58), a protein gradient was not observed for either channel in this study. Perhaps this is because the tissue sections employed in this study were from the left ventricular free wall base and the gradient is more apparent in the middle and apex of the ventricle (6). Together, these immunostaining data argue for altered expression in the left ventricular myocytes as opposed to cells derived from, for example, vascular beds.


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  		Fig. 4.   Immunodetection of relative Kv4.2 and Kv1.4 protein levels in diabetic ventricle. A and B: staining of anti-Kv4.2 antiserum to C and DM rat heart, respectively. C and D: staining to control tissue with either no primary antibody or after preincubation of the primary antibody with the peptide immunogen. E and F: staining of affinity-purified anti-Kv4.2 antibody to C and DM rat heart, respectively. G and H: staining of affinity-purified anti-Kv1.4 antibody to C and DM rat heart, respectively.

Western blot analysis of Kv4.2 and Kv1.4 protein levels in diabetic ventricle. We also confirmed via Western blot analysis that Kv4.2 and Kv1.4 protein levels in the ventricle changed in parallel to the observed changes in mRNA. As shown in Fig. 5, Kv1.4 protein was dramatically increased with the diabetic state, whereas Kv4.2 was markedly reduced. Because of the limited depth of film, quantitation was not attempted. Still, these data confirm the mRNA analysis and thus further support the idea that the change from ITOfast to ITOslow accompanying diabetes is due to the upregulation of the Kv1.4 protein expression at the expense of Kv4.2 channel expression.


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  		Fig. 5.   Western blot analysis of Kv1.4 and Kv4.2 protein expression in DM and C ventricle. A: immunodetection of Kv1.4 protein in representative DM and C samples, respectively. B: levels of Kv4.2 protein. Equal amounts of membrane protein were loaded in each lane as confirmed by Ponceau staining and analysis with antitubulin antibodies (data not shown). Arrows, position of Kv1.4 and Kv4.2alpha subunits.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Abnormal Kv gene expression in diabetes. APD is prolonged in the STZ-induced diabetic rat ventricle (31, 38), and the attenuation of ITO has been proposed as being responsible (48-50). Furthermore, an increased rate dependency (38) or prolongation of recovery kinetics of ITO was reported (49, 50). When heterologously expressed, Kv1.4 and Kv4.2 produce rapidly activating and inactivating outward currents and are considered to underlie cardiac ITO (9, 51, 55, 63). One of the greatest differences between these Kv channels is their recovery from inactivation. Kv1.4 recovers with a time constant of 3-8 s (2, 40, 41), whereas the Kv4.2 recovers much faster with a time constant of 200-1,000 ms (57, 58). Recent gene knockout and dominant negative approaches have confirmed that ITOfast is encoded by Kv4.2, whereas ITOslow is due to Kv1.4 (19, 62).

The data shown above indicate that Kv4.2 gene expression (Fig. 2B) and protein (Fig. 4, A and B, and Fig. 5) levels were reduced, whereas Kv1.4 gene expression (Fig. 2A) and protein levels (Fig. 4, G and H, and Fig. 5) were increased in diabetic heart ventricles. These changes are likely to represent the molecular mechanism responsible for the effects of diabetes on ITO and the resulting increase in cardiac APD. Our findings support recently published work of Qin et al. (42), which shows that diabetes reduces ventricular Kv4.2 protein and mRNA levels. However, this study did not examine the levels of either Kv1.4 mRNA or protein. In contrast to our findings reported here, these investigators found Kv4.3 expression was also suppressed with diabetes.

Interestingly, Kv4.2 and Kv1.4 gene expression was changed without marked cardiac hypertrophy (Table 1, Fig. 1) (42), albeit MHC gene expression was abnormal. This shows that the alteration in Kv4.2 and Kv1.4 mRNA levels was not secondary to ventricular hypertrophy. K+ channel expression in the heart is associated with cardiac hypertrophy (29), and it seems likely we would also have observed hypertrophy with longer duration or more deeply diabetic rats. The abnormal Kv1.4 and Kv4.2 gene expression in the ventricle was not observed in the brain (Fig. 3, A and B), showing that this abnormality could not be simply ascribed to hyperglycemia. Other possibilities include perhaps hemodynamic factors (12, 27, 56) or impaired autonomic neural control of the heart (17, 30, 53). In fact, the heart rate of the STZ-induced diabetic rats is reduced (12, 27, 56). It is interesting that, although both Kv4.2 and Kv4.3 contribute to cardiac ITO, only the Kv4.2 expression was altered.

Our present data are in contrast to several other studies examining gene expression in hypertensive hearts or after infarction. Takimoto et al. (51) showed that in renovascular hypertensive rats, both Kv4.2 and Kv4.3 expression were decreased, whereas Kv1.4 expression was unaltered. In one study examining altered ITO expression after infarction both Kv4.2 and Kv4.3 were downregulated, whereas Kv1.4 expression increased (26).

Clearly, the cardiac K+ gene expression responds differently to hypertension, infarction, and the diabetic state studied in our present work. IGF treatment of diabetic rats did not affect the changes in Kv channel expression in the ventricle or brain. This treatment also had no effect on these tissues in nondiabetic rats.

Low-dose IGF treatment protects against MHC isoform switching. alpha -MHC mRNA was reduced 32% (Fig. 1A), whereas beta -MHC was increased 259% (Fig. 1B) in diabetic ventricles. This isoform switching is observed in cardiac but not skeletal muscle (35). It is noteworthy that MHC isoform switching was prevented by low-dose (~15 µg · kg-1 · day-1) IGF-II treatment, despite continued hyperglycemia and body weight loss (Table 1). This suggests that isoform switching may not be a consequence of hyperglycemia per se but may result from reduced IGF gene expression in the heart (5, 28). Likewise, low-dose IGF-I or IGF-II treatment can prevent diabetic neuropathy independently of hyperglycemia and weight loss (22, 64). Further study is needed to determine whether IGF protection results from indirect actions on autonomic function or direct actions on cardiac tissue. IGF treatment both prevents ultrastructural abnormalities in autonomic nerves (47) and attenuates myocardial injury and apoptosis after ischemia (7). Thus IGF-I improves cardiac output and decreases systemic vascular resistance after infarction (10). Both IGF-I and IGF-II act through the type I IGF receptor expressed on the surface of ventricular myocytes (18).

In summary, we have demonstrated that K+ channel genes regulating the ITO switch from Kv4.2 to Kv1.4 in diabetic ventricles. This change may well explain the reduced rate of repolarization and abnormal ITO observed in the diabetic heart. The switch in Kv channel isoform expression preceded observable cardiac hypertrophy, albeit MHC isoform gene expression was altered. IGF treatment blunted the diabetes-induced changes in MHC isoform gene expression independently of continued hyperglycemia but had no effect on Kv channel expression. These findings suggest IGF treatment may ameliorate some but not all aspects of cardiac contractile dysfunction in diabetes.


    ACKNOWLEDGEMENTS

This research was supported by National Heart, Lung, and Blood Institute Grant RO1-HL-49330 (to M. M. Tamkun), National Institute of Diabetes and Digestive and Kidney Diseases Grant RO1-DK-53922 (to D. N. Ishii), and a fellowship from Medtronix Japan (to A. Nishiyama).


    FOOTNOTES

Address for reprint requests and other correspondence: M. M. Tamkun, Dept. of Physiology, Colorado State Univ., Ft. Collins, CO, 80523 (tamkunmm{at}lamar.colostate.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.

Received 27 April 2000; accepted in final form 2 July 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Ausubel, FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, and Strul K (Editors). Current Protocols in Molecular Biology. New York: Wiley, 1989.

2.   Bertoli, A, Moran O, and Conti F. Activation and deactivation properties of rat brain K+ channels of the Shaker-related subfamily. Eur Biophys J 23: 379-784, 1994[ISI][Medline].

3.   Blair, TA, Roberds SL, Tamkun MM, and Hartshorne RP. Functional expression of RK5, a voltage-gated K+ channel isolated from the rat cardiovascular system. FEBS Lett 295: 211-213, 1991[ISI][Medline].

4.   Bornfeldt, KE, Arnqvist HJ, Enberg B, Mathews LS, and Norstedt G. Regulation of insulin-like growth factor-I and growth hormone receptor gene expression by diabetes and nutritional state in rat tissues. J Endocrinol 122: 651-656, 1989[Abstract].

5.   Bornfeldt, KE, Skottner A, and Arnqvist HJ. In-vivo regulation of messenger RNA encoding insulin-like growth factor-I (IGF-I) and its receptor by diabetes, insulin and IGF-I in rat muscle. J Endocrinol 135: 203-211, 1992[Abstract].

6.   Brahmajothi, MV, Campbell DL, Rasmusson RL, Morales MJ, Trimmer JS, Nerbonne JM, and Strauss HC. Distinct transient outward potassium current (Ito) phenotypes and distribution of fast-inactivating potassium channel alpha subunits in ferret left ventricular myocytes. J Gen Physiol 113: 581-600, 1999[Abstract/Free Full Text].

7.   Buerke, M, Murohara T, Skurk C, Nuss C, Tomaselli K, and Lefer AM. Cardioprotective effect of insulin-like growth factor I in myocardial ischemia followed by reperfusion. Proc Natl Acad Sci USA 92: 8031-8035, 1995[Abstract/Free Full Text].

8.   Dixon, JE, and McKinnon D. Quantitative analysis of potassium channel mRNA expression in atrial and ventricular muscle of rats. Circ Res 75: 252-260, 1994[Abstract/Free Full Text].

9.   Dixon, JE, Shi W, Wang HS, McDonald C, Yu H, Wymore RS, Cohen IS, and McKinnon D. Role of the Kv4.3 K+ channel in ventricular muscle A molecular correlate for the transient outward current. Circ Res 79: 659-668, 1996[Abstract/Free Full Text].

10.   Duerr, RL, McKirnan MD, Gim RD, Clark RG, Chien KR, and Ross J, Jr. Cardiovascular effects of insulin-like growth factor-1 and growth hormone in chronic left ventricular failure in the rat. Circulation 93: 2188-2196, 1996[Abstract/Free Full Text].

11.   Fagin, JA, Roberts CT, Jr, LeRoith D, and Brown AT. Coordinate decrease of tissue insulinlike growth factor I posttranscriptional alternative mRNA transcripts in diabetes mellitus. Diabetes 38: 428-434, 1989[Abstract].

12.   Fazan, R, Jr, Ballejo G, Salgado MC, Moraes MF, and Salgado HC. Heart rate variability and baroreceptor function in chronic diabetic rats. Hypertension 30: 632-635, 1997[Abstract/Free Full Text].

13.   Fein, FS. Diabetic cardiomyopathy. Diabetes Care 13: 1169-1179, 1990[Abstract].

14.   Fein, FS, Aronson RS, Nordin C, Miller-Green B, and Sonnenblick EH. Altered myocardial response to ouabain in diabetic rats: mechanics and electrophysiology. J Mol Cell Cardiol 15: 769-784, 1983[ISI][Medline].

15.   Fein, FS, Kornstein LB, Strobeck JE, Capasso JM, and Sonnenblick EH. Altered myocardial mechanics in diabetic rats. Circ Res 47: 922-933, 1980[Abstract/Free Full Text].

16.   Fein, FS, and Sonnenblick EH. Diabetic cardiomyopathy. Prog Cardiovasc Dis 27: 255-270, 1985[ISI][Medline].

17.   Felten, SY, Peterson RG, Shea PA, Besch HR, Jr, and Felten DL. Effects of streptozotocin diabetes on the noradrenergic innervation of the rat heart: a longitudinal histofluorescence and neurochemical study. Brain Res Bull 8: 593-607, 1982[ISI][Medline].

18.   Foncea, R, Andersson M, Ketterman A, Blakesley V, Sapag-Hagar M, Sugden PH, LeRoith D, and Lavandero S. Insulin-like growth factor-I rapidly activates multiple signal transduction pathways in cultured rat cardiac myocytes. J Biol Chem 272: 19115-19124, 1997[Abstract/Free Full Text].

19.   Guo, W, Xu H, London B, and Nerbonne JM. Molecular basis of transient outward K+ current diversity in mouse ventricular myocytes. J Physiol (Lond) 521: 587-599, 1999[Abstract/Free Full Text].

20.   Horackova, M, and Murphy MG. Effects of chronic diabetes mellitus on the electrical and contractile activities, 45Ca2+ transport, fatty acid profiles and ultrastructure of isolated rat ventricular myocytes. Pflügers Arch 411: 564-572, 1988[ISI][Medline].

21.   Ishii, DN. Implication of insulin-like growth factors in the pathogenesis of diabetic neuropathy. Brain Res Brain Res Rev 20: 47-67, 1995[Medline].

22.   Ishii, DN, and Lupien SB. Insulin-like growth factors protect against diabetic neuropathy: effects on sensory nerve regeneration in rats. J Neurosci Res 40: 138-144, 1995[ISI][Medline].

23.   Izumo, S, Nadal-Ginard B, and Mahdavi V. All members of the MHC multigene family respond to thyroid hormone in a highly tissue-specific manner. Science 231: 597-600, 1986[Abstract/Free Full Text].

24.   Jourdon, P, and Feuvray D. Calcium and potassium currents in ventricular myocytes isolated from diabetic rats. J Physiol (Lond) 470: 411-429, 1993[Abstract/Free Full Text].

25.   Kannel, WB, and McGee DL. Diabetes and cardiovascular disease. The Framingham study. JAMA 241: 2035-2038, 1979[Abstract].

26.   Kaprielian, R, Wickenden AD, Kassiri Z, Parker TG, Liu PP, and Backx PH. Relationship between K+ channel down-regulation and [Ca2+]i in rat ventricular myocytes following myocardial infarction. J Physiol (Lond) 517: 229-245, 1999[Abstract/Free Full Text].

27.   Kato, K, Chapman DC, Rupp H, Lukas A, and Dhalla NS. Alterations of heart function and Na+-K+-ATPase activity by etomoxir in diabetic rats. J Appl Physiol 86: 812-818, 1999[Abstract/Free Full Text].

28.   Leaman, DW, Simmen FA, Ramsay TG, and White ME. Insulin-like growth factor-I and -II messenger RNA expression in muscle, heart, and liver of streptozotocin-diabetic swine. Endocrinology 126: 2850-2857, 1990[Abstract].

29.   Levitan, ES, and Takimoto K. Dynamic regulation of K+ channel gene expression in differentiated cells. J Neurobiol 37: 60-68, 1998[ISI][Medline].

30.   Lund, DD, Subieta AR, Pardini BJ, and Chang KS. Alterations in cardiac parasympathetic indices in STZ-induced diabetic rats. Diabetes 41: 160-166, 1992[Abstract].

31.   Magyar, J, Rusznak Z, Szentesi P, Szucs G, and Kovacs L. Action potentials and potassium currents in rat ventricular muscle during experimental diabetes. J Mol Cell Cardiol 24: 841-853, 1992[ISI][Medline].

32.   Malhotra, A, Penpargkul S, Fein FS, Sonnenblick EH, and Scheuer J. The effect of streptozotocin-induced diabetes in rats on cardiac contractile proteins. Circ Res 49: 1243-1250, 1981[Abstract/Free Full Text].

33.   Martens, JR, Navarro-Polanco R, Coppock EA, Nishiyama A, Parshley L, Grobaski TD, and Tamkun MM. Differential targeting of shaker-like potassium channels to lipid rafts. J Biol Chem 275: 7443-7446, 2000[Abstract/Free Full Text].

34.   Mays, DJ, Foose JM, Philipson LH, and Tamkun MM. Localization of the Kv1.5 K+ channel protein in explanted cardiac tissue. J Clin Invest 96: 282-292, 1995.

35.   Morris, GS, Prevost MC, and Nelson AG. Moderate diabetes alters myosin isoenzyme distribution in cardiac but not skeletal muscle of male rats. Life Sci 58: 833-838, 1996[ISI][Medline].

36.   Nishiyama, A, Kambe F, Kamiya K, Seo H, and Toyama J. Effects of thyroid status on expression of voltage-gated potassium channels in rat left ventricle. Cardiovasc Res 40: 343-351, 1998[Abstract/Free Full Text].

37.   Nobe, S, Aomine M, Arita M, Ito S, and Takaki R. Chronic diabetes mellitus prolongs action potential duration of rat ventricular muscles: circumstantial evidence for impaired Ca2+ channel. Cardiovasc Res 24: 381-389, 1990[Abstract/Free Full Text].

38.   Pacher, P, Ungvari Z, Nanasi PP, and Kecskemeti V. Electrophysiological changes in rat ventricular and atrial myocardium at different stages of experimental diabetes. Acta Physiol Scand 166: 7-13, 1999[ISI][Medline].

39.   Phillips, LS, and Young HS. Nutrition and somatomedin. II. Serum somatomedin activity and cartilage growth activity in streptozotocin-diabetic rats. Diabetes 25: 516-527, 1976[Abstract].

40.   Po, S, Roberds S, Snyders DJ, Tamkun MM, and Bennett PB. Heteromultimeric assembly of human potassium channels. Molecular basis of a transient outward current? Circ Res 72: 1326-1336, 1993[Abstract/Free Full Text].

41.   Po, S, Snyders DJ, Baker R, Tamkun MM, and Bennett PB. Functional expression of an inactivating potassium channel cloned from human heart. Circ Res 71: 732-736, 1992[Abstract/Free Full Text].

42.   Qin, D, Huang B, Deng L, El-Adawi H, Ganguly K, Sowers JR, and El-Sherif N. Downregulation of K(+) channel genes expression in type I diabetic cardiomyopathy. Biochem Biophys Res Commun 283: 549-553, 2001[ISI][Medline].

43.   Regan, TJ, Lyons MM, Ahmed SS, Levinson GE, Oldewurtel HA, Ahmad MR, and Haider B. Evidence for cardiomyopathy in familial diabetes mellitus. J Clin Invest 60: 884-899, 1977.

44.   Ren, J, and Davidoff AJ. Diabetes rapidly induces contractile dysfunctions in isolated ventricular myocytes. Am J Physiol Heart Circ Physiol 272: H148-H158, 1997[Abstract/Free Full Text].

45.   Rodrigues, B, and McNeill JH. Cardiac function in spontaneously hypertensive diabetic rats. Am J Physiol Heart Circ Physiol 251: H571-H580, 1986.

46.   Schiaffino, S, Samuel JL, Sassoon D, Lompre AM, Garner I, Marotte F, Buckingham M, Rappaport L, and Schwartz K. Nonsynchronous accumulation of alpha -skeletal actin and beta -myosin heavy chain mRNAs during early stages of pressure-overload-induced cardiac hypertrophy demonstrated by in situ hybridization. Circ Res 64: 937-948, 1989[Abstract/Free Full Text].

47.   Schmidt, RE, Dorsey DA, Beaudet LN, Plurad SB, Parvin CA, and Miller MS. Insulin-like growth factor I reverses experimental diabetic autonomic neuropathy. Am J Pathol 155: 1651-1660, 1999[Abstract/Free Full Text].

48.   Shimoni, Y, Ewart HS, and Severson D. Type I and II models of diabetes produce different modifications of K+ currents in rat heart: role of insulin. J Physiol (Lond) 507: 485-496, 1998[Abstract/Free Full Text].

49.   Shimoni, Y, Firek L, Severson D, and Giles W. Short-term diabetes alters K+ currents in rat ventricular myocytes. Circ Res 74: 620-628, 1994[Abstract/Free Full Text].

50.   Shimoni, Y, Severson D, and Giles W. Thyroid status and diabetes modulate regional differences in potassium currents in rat ventricle. J Physiol (Lond) 488: 673-688, 1995[ISI][Medline].

51.   Takimoto, K, Li D, Hershman KM, Li P, Jackson EK, and Levitan ES. Decreased expression of Kv4.2 and novel Kv4.3 K+ channel subunit mRNAs in ventricles of renovascular hypertensive rats. Circ Res 81: 533-539, 1997[Abstract/Free Full Text].

52.   Tan, K, and Baxter RC. Serum insulin-like growth factor I levels in adult diabetic patients: the effect of age. J Clin Endocrinol Metab 63: 651-655, 1986[Abstract].

53.   Tomlinson, DR, and Yusof AP. Autonomic neuropathy in the alloxan-diabetic rat. J Auton Pharmacol 3: 257-263, 1983[ISI][Medline].

54.   Tomlinson, KC, Gardiner SM, Hebden RA, and Bennett T. Functional consequences of streptozotocin-induced diabetes mellitus, with particular reference to the cardiovascular system. Pharmacol Rev 44: 103-150, 1992[ISI][Medline].

55.   Tseng, GN. Molecular structure of cardiac Ito channels: Kv4.2, Kv4.3, and other possibilities. Cardiovasc Res 41: 16-18, 1999[Free Full Text].

56.   Van Buren, T, Kasbergen CM, Gispen WH, and De Wildt DJ. In vivo cardiovascular reactivity and baroreflex activity in diabetic rats. Cardiovasc Res 38: 763-771, 1998[Abstract/Free Full Text].

57.   Wang, HS, Dixon JE, and McKinnon D. Unexpected and differential effects of Cl- channel blockers on the Kv4.3 and Kv4.2 K+ channels Implications for the study of the I(to2) current. Circ Res 81: 711-718, 1997[Abstract/Free Full Text].

58.   Wickenden, AD, Jegla TJ, Kaprielian R, and Backx PH. Regional contributions of Kv1.4, Kv4.2, and Kv4.3 to transient outward K+ current in rat ventricle. Am J Physiol Heart Circ Physiol 276: H1599-H1607, 1999[Abstract/Free Full Text].

59.   Winter, RJ, Plotnick LP, and Thompson RG. Chemical diabetes in childhood. Integrated concentrations of glucose, insulin, and growth hormone. Diabetes 27: 909-915, 1978[Abstract].

60.   Wuarin, L, Guertin DM, and Ishii DN. Early reduction in insulin-like growth factor gene expression in diabetic nerve. Exp Neurol 130: 106-114, 1994[ISI][Medline].

61.   Xu, H, Dixon JE, Barry DM, Trimmer JS, Merlie JP, McKinnon D, and Nerbonne JM. Developmental analysis reveals mismatches in the expression of K+ channel alpha subunits and voltage-gated K+ channel currents in rat ventricular myocytes. J Gen Physiol 108: 405-419, 1996[Abstract/Free Full Text].

62.   Xu, H, Li H, and Nerbonne JM. Elimination of the transient outward current and action potential prolongation in mouse atrial myocytes expressing a dominant negative Kv4 alpha subunit. J Physiol (Lond) 519: 11-21, 1999[Abstract/Free Full Text].

63.   Xu, Z, Patel KP, and Rozanski GJ. Metabolic basis of decreased transient outward K+ current in ventricular myocytes from diabetic rats. Am J Physiol Heart Circ Physiol 271: H2190-H2196, 1996[Abstract/Free Full Text].

64.   Zhuang, HX, Wuarin L, Fei ZJ, and Ishii DN. Insulin-like growth factor (IGF) gene expression is reduced in neural tissues and liver from rats with noninsulin-dependent diabetes mellitus, and IGF treatment ameliorates diabetic neuropathy. J Pharmacol Exp Ther 283: 366-374, 1997[Abstract/Free Full Text].

Am J Physiol Heart Circ Physiol 281(4):H1800-H1807
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