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Am J Physiol Heart Circ Physiol 278: H1105-H1116, 2000;
0363-6135/00 $5.00
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Vol. 278, Issue 4, H1105-H1116, April 2000

The thyroid hormone analog DITPA restores Ito in rats after myocardial infarction

Alan D. Wickenden, Roger Kaprielian, Xiao-Mang You, and Peter H. Backx

Departments of Physiology and Medicine and The Center for Cardiovascular Research, University Health Network, University of Toronto, Toronto, Ontario, Canada M5G 2C4


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Previous studies have established that reductions in repolarizing currents occur in heart disease and can contribute to life-threatening arrhythmias in myocardium. In this study, we investigated whether the thyroid hormone analog 3,5-diiodothyropropionic acid (DITPA) could restore repolarizing transient outward K+ current (Ito) density and gene expression in rat myocardium after myocardial infarction (MI). Our findings show that Ito density was reduced after MI (14.0 ± 1.0 vs. 10.2 ± 0.9 pA/pF, sham vs. post-MI at +40 mV). mRNA levels of Kv4.2 and Kv4.3 genes were decreased but Kv1.4 mRNA levels were increased post-MI. Corresponding changes in Kv4.2 and Kv1.4 protein were also observed. Chronic treatment of post-MI rats with 10 mg/kg DITPA restored Ito density (to 15.2 ± 1.1 pA/pF at +40 mV) as well as Kv4.2 and Kv1.4 expression to levels observed in sham-operated controls. Other membrane currents (Na+, L-type Ca2+, sustained, and inward rectifier K+ currents) were unaffected by DITPA treatment. Associated with the changes in Ito expression, action potential durations (current-clamp recordings in isolated single right ventricular myocytes and monophasic action potential recordings from the right free wall in situ) were prolonged after MI and restored with DITPA treatment. Our results demonstrate that DITPA restores Ito density in the setting of MI, which may be useful in preventing complications associated with Ito downregulation.

action potential; transient outward current; Kv4.2; Kv4.3; Kv1.4; 3,5-diiodothyropropionic acid


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

VENTRICULAR REMODELING is a well-recognized response to myocyte loss such as occurs after a myocardial infarction (MI) (33). The remodeling process involves concentric myocyte hypertrophy and altered gene expression in surviving myocytes in both left and right ventricles (1, 7, 37, 48). An important feature of the remodeling process is action potential prolongation secondary to reductions in the transient outward K+ current (Ito) density, which can increase intracellular Ca2+ concentration ([Ca2+]i) transient amplitude (21) and thus enhance contractility of the compromised heart (44). On the other hand, chronic elevations of [Ca2+]i associated with action potential prolongation may potentiate hypertrophic signaling cascades and lead to maladaptive gene expression, thereby overriding any short-term benefits and perhaps contributing to left ventricular dysfunction and failure (44). Action potential prolongation can also be arrhythmogenic, and, as such, this compensatory mechanism may predispose the heart to arrhythmias and sudden cardiac death (40).

Given the potential contribution of Ito downregulation and action potential prolongation to the pathogenesis of heart failure, normalization of Ito in the hypertrophied heart might be useful in the treatment of this condition. In this regard, thyroid hormone can increase the expression of genes encoding for Ito (17, 39, 45), alter the biophysical properties of Ito (38, 39, 45), and abbreviate action potential duration (4). The effects of thyroid hormone, however, are complicated by a plethora of noncardiac effects that may limit the beneficial effects of this agent (14). In this study, we chose to examine the effects of the thyroid hormone analog 3,5-diiodothyropropionic acid (DITPA) on Ito expression in the postinfarcted rat heart, because this analog binds to nuclear thyroid hormone receptors and can alter the transcription of triiodothyronine-responsive genes (31) with apparently only minor effects on heart rate and metabolism compared with thyroid hormone itself (25, 32). Our results show that chronic treatment of infarcted hearts with DITPA restored Ito expression and hastened repolarization to levels observed in noninfarcted hearts. A preliminary report of our data has appeared (43).


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

Left anterior descending coronary artery ligation procedure. The left anterior descending (LAD) ligation procedure was performed on male Lewis-Brown Norwegian (LBN-F1 strain, Harlan, Indianapolis, IN) rats (220 g, 10-12 wk of age) as previously described (30). Briefly, animals were anesthetized with ketamine hydrochloride (45 mg/kg) and xylazine (5 mg/kg) intraperitoneally. Once an adequate depth of anesthesia was achieved, animals were intubated with a 14-gauge polyethylene (PE) catheter and ventilated with room air using a small animal ventilator (model 683, Harvard Apparatus, Boston, MA). A left thoracotomy was performed in the fifth intercostal space, and the pericardium was opened. The proximal left coronary artery under the tip of the left atrial appendage was encircled and ligated using a 6-0 silk suture. The muscle and skin were closed in layers. For sham-operated animals, the left coronary artery was identified and encircled but not ligated, and the muscle and skin layers were closed similarly. The experimental and animal care protocol was approved by the Committee on Animal Research at the Toronto General Hospital and was performed in accordance with the "Position of the American Heart Association on Research Animal Use." After the surgical procedure was completed, rats were housed in pairs in a climate-controlled environment at an ambient temperature of 21°C with a 12:12-h light-dark cycle and were given water and standard rat chow ad libitum.

Five weeks after the LAD ligation procedure was completed, animals were treated with either DITPA (3.75 or 10 mg/kg body wt) or NaCl (0.9 g/100 ml). Stock solutions containing 3.75 or 10 mg/ml DITPA (Sigma Chemical, St. Louis, MO) were prepared as previously described (32) and diluted in 0.9 g/100 ml NaCl. The doses of DITPA used in the present study were previously shown to exert beneficial effects on contractile performance in animal models of cardiac hypertrophy and failure (25, 32). Animals received daily subcutaneous injections for 21 days. Eight weeks after the sham or LAD ligation procedure was completed, the animals were killed and mRNA levels, protein levels, current densities, and action potentials were measured using the methods described below. In addition, the left ventricular free wall was carefully dissected from base to apex and along the border edge of the right ventricle. Infarct size was determined by measuring the infarcted area on the epicardial surface and was expressed as a percentage of the area of the left ventricular free wall.

The right ventricle was chosen for these studies because myocyte hypertrophy and the altered gene expression that occurs in the right ventricle after left-sided infarctions (1, 3, 37, 48) may be important prognostic determinants in heart disease (49). Furthermore, isolation of myocytes from the right ventricle avoids potential problems associated the presence of a large scar in the infarcted left ventricle. We chose to make our recordings 8 wk post-MI because previous characterization of this model established that this time point represents a phase of compensated hypertrophy, rather than failure (15), and would encompass the period during which there is an increased incidence of arrhythmias (35). Therefore, 8 wk postinfarction is a relevant time point at which to study mechanisms with potential relevance to hypertrophy and arrhythmogenesis.

Monophasic action potential and hemodynamic studies. For monophasic action potential recordings, isolated hearts were retrogradely perfused with a solution containing (in mM) 140 NaCl, 5.4 KCl, 10 HEPES, 1 MgCl2, and 10 D-glucose, with pH balanced to 7.4 with NaOH at 37°C. Ca2+ was excluded from this solution to eliminate possible artifacts associated with wall motion. A custom-made contact probe was used to record monophasic action potentials as previously described (12). The monophasic action potential probe consisted of a bipolar electrode (0.8 mm in diameter) of sintered Ag-AgCl pellet (E255, In Vivo Metric, Healdsburg, CA) encased in PE tubing (with a total outer diameter of 1.3 mm). The monophasic action potential probe was gently pressed against the centrolateral surface of the right ventricular wall with constant pressure to eliminate electrical artifacts associated with slippage or uneven contact (13). Monophasic action potentials were filtered at 1 kHz and recorded at 10 kHz.

For hemodynamic assessment, animals were anesthetized with ketamine hydrochloride (45 mg/kg) and xylazine (5 mg/kg) injected intraperitoneally. The right carotid artery was cannulated with PE-200 tubing that was drawn to a diameter of ~100-200 µm. The tubing was attached to a pressure transducer to record hydrostatic pressure. The catheter was advanced into the aorta and then into the left ventricle. From the pressure traces, left ventricular end-diastolic (LVEDP) and end-systolic pressure (LVESP) as well as the maximal derivatives of pressure (i.e., +dP/dt and -dP/dt) were measured.

Isolation of right ventricular myocytes. The procedure used for isolation of rat ventricular myocytes was described in detail previously (21). Heparinized (500 U ip) rats were anesthetized with pentobarbital sodium (75 mg/kg ip). Once an adequate depth of anesthesia was achieved, the hearts were quickly removed and retrogradely perfused for ~3 min with a Ca2+-containing Tyrode solution of the following composition (in mM): 140 NaCl, 5.4 KCl, 10 HEPES, 1 MgCl2, 1 CaCl2, and 10 D-glucose, with pH balanced to 7.4 with NaOH at 37°C. The heart was then perfused with Ca2+-free Tyrode solution for 5 min before the heart was digested with the same solution containing collagenase (type II, 0.55 mg/ml, Boehringer-Mannheim) and protease (type XIV, 0.05 mg/ml, Sigma Chemical) for 8-9 min. The enzyme solution was subsequently removed by perfusion with a Kraftbrühe (high K+) solution containing (in mM) 120 K-glutamate, 20 KCl, 20 HEPES, 1 MgCl2, 0.3 K-EGTA, and 10 D-glucose for 5 min. All solutions were prebubbled with 100% O2 for 5 min. After the enzyme washout period, the atria and blood vessels were removed and the ventricles were separated. The right ventricular free wall was dissected free from the remainder of the ventricle. Myocytes were minced and mechanically agitated in a high-K+ solution containing BSA (0.02% wt/vol). Myocytes were then filtered through a nylon mesh and resuspended in a Kraftbrühe solution with 50 mg/ml gentamicin. Ca2+-tolerant, quiescent rod-shaped cells with clear, regular cross-striations were selected for electrophysiological recordings. Cells were transferred into a perfusion bath situated on the stage of an inverted microscope and perfused with recording solution at a rate of 1-2 ml/min.

Electrophysiological measurements in right ventricular myocytes. Current densities [Ito, inward rectifier K+ current (IK1), sustained current (Isus), Na+ current (INa), L-type Ca2+ current (ICa,L)] and action potentials were measured using the whole cell patch-clamp recording method (18) under voltage- and current-clamp conditions with an Axopatch 200A amplifier (Axon Instruments, Foster City, CA) as described in detail previously (21). When pipettes were filled with intracellular solution, pipette tip resistances were typically 2-3 MOmega . Series resistance compensation ranged between 60 and 90%. After membrane rupture, we estimated the cell capacitance by integrating the area of the capacitance transients after a 5-mV step from a holding potential of -80 mV.

For K+ currents, myocytes were superfused with a standard Tyrode solution containing (in mM) 140 NaCl, 1 MgCl2, 10 HEPES, 4 KCl, 1 CaCl2, and 10 D-glucose, with pH adjusted to 7.4 with NaOH. Intracellular solutions for K+ currents and action potentials contained (in mM) 130 K-aspartate, 20 KCl, 5 EGTA, 5 NaCl, 10 HEPES, and 5 MgATP, with pH adjusted to 7.2 with Trizma base. Action potentials were also recorded with an intracellular solution containing 30 mM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) and a modified Tyrode solution containing 0.1 mM CaCl2. Recordings were not corrected for a liquid junction potential of approximately -8 mV.

To isolate Ito, we used prepulses to -40 mV for 30 ms to inactivate INa, and 0.3 mM CdCl2 was added to block ICa,L. Although CdCl2 has been reported to shift Ito channel gating (2, 46), it was the preferred choice to block ICa,L because organic Ca2+ channel blockers have been shown to affect Ito amplitude and kinetics (22). Ito was defined as the peak current (Ipeak) elicited by 500-ms voltage steps in the range from -30 to +70 mV minus the steady-state current (Isus) remaining at the end of the 500-ms voltage step.

To measure IK1, we elicited a series of 500-ms steps from -130 to 0 mV (in 10-mV increments) in the presence and absence of 0.3 mM BaCl2. IK1 was defined as the Ba2+-sensitive current component. To measure ICa,L, we replaced the KCl in the Tyrode solution with equimolar CsCl while intracellular solutions contained (in mM) 150 CsCl, 10 HEPES, 1 MgCl2, 5 EGTA and 5 MgATP, with pH adjusted to 7.2 with CsOH. A brief prepulse to -40 mV for 100 ms was used to inactivate INa. ICa,L was estimated as peak Cd2+-sensitive current in response to 500-ms voltage steps from -60 to +70 mV. To measure INa, we superfused myocytes with a solution containing (in mM) 5 NaCl, 125 triethylamine hydrochloride (TEA-Cl), 1 MgCl2, 20 HEPES, 5 CsCl, 1 MnCl2, and 10 D-glucose, with pH adjusted to 7.4 with CsOH. Intracellular solution contained (in mM) 125 CsCl, 20 TEA-Cl, 10 HEPES, 10 EGTA, and 5 MgATP, with pH adjusted to 7.2 with CsOH. A series of 50-ms voltage steps was applied between -80 and +10 mV in 5-mV increments from a holding potential of -100 mV. A P/4 protocol was used for leak and capacitance subtraction in the INa experiments. All experiments were performed at room temperature (19-21°C).

Preparation of total RNA from the right ventricle and RNase protection assays. Rat right ventricles and brains were isolated, rinsed briefly in a standard Tyrode solution, and snap-frozen in liquid N2. Ventricular tissue was powdered and RNA extracted by the one-step acid guanidinium-phenol method (8). The concentration of RNA was measured spectrophotometrically and confirmed by agarose gel electrophoresis. Gels were loaded with 10 µg of total RNA obtained from three hearts per group. RNase protection assays were performed using an RPAII Ribonuclease Protection Assay Kit (Ambion, Austin, TX) as previously described (21, 42, 45). The Kvx probes for RNase protection assays were kindly provided by Dr. David McKinnon (State University of New York at Stony Brook, Stony Brook, NY) and have been described previously (10, 11, 42). The cyclophilin probe was purchased from Ambion. The myosin heavy chain (MHC) probe was kindly supplied by Drs. J. N. Tsoporis and T. G Parker (University of Toronto, Toronto, ON, Canada) and was also described previously (41). Abundance of mRNA transcripts was quantified by densitometry (Bio-Rad GS670 Imaging densitometer). Signals were normalized to a cyclophilin internal standard to ensure that findings were not influenced by minor variations in loading.

Isolation of protein from the right ventricle. Rat right ventricles or brains from three rats per group were quickly rinsed, frozen in liquid N2, and stored at -70°C before isolation of membrane proteins. Frozen tissue was homogenized in 10 volumes of 0.3 M sucrose and 30 mM histidine, and the resulting homogenate was then centrifuged at 3,000 g for 15 min. The supernatant was collected and recentrifuged at 45,000 g for 1 h to precipitate membrane protein. The membrane pellet was resuspended in 1% Triton X-100 and 50 mM Tris (pH 6.8), left on ice for 1 h, and then centrifuged at 14,000 g for 15 min. The final supernatant was saved for protein assay by the Lowry method, and Western blot analysis was performed. All solutions in this procedure were chilled on ice and contained protease inhibitors (0.1 mM phenylmethylsulfonyl fluoride and 5 µg/ml each of aprotinin, leupeptin, antipain, and pepstatin).

Preparation of anti-Kv1.4 and anti-Kv4.2 antibodies. Anti-Kv1.4 and anti-Kv4.2 antibodies were kindly supplied by Dr. O. T. Jones (Department of Pharmacology, University of Toronto). The procedure by which the anti-Kv1.4 and anti-Kv4.2 antibodies were produced was described in detail previously (42, 45).

Western blot analysis. For Western blot analysis, 50-100 µg of total heart protein and 10-20 µg of total brain protein were resolved on a 10% SDS-PAGE gel and transferred to a polyvinylidene fluoride membrane. After the transfer, the membrane was rinsed with Tris-buffered saline (TBS; 150 mM NaCl and 20 mM Tris, pH 7.5). Blots were blocked with 10% Carnation Instant milk powder in TBS for 1 h and probed with anti-Kv antibodies diluted in 3% milk-TBS overnight at 4°C. After being washed with TBS to remove excess primary antibody, the blots were incubated with secondary antibody (donkey anti-rabbit IgG conjugated to horseradish peroxidase, Amersham) in blocking buffer for 1 h. The membrane was washed again with TBS containing 0.05% Tween 20 and 1% Triton X-100 and developed by enhanced chemiluminescence (ECL reagent, Amersham). Gel loading was checked by staining total proteins with Ponceau S, and molecular weights were determined using prestained markers (Kaleidoscope, Bio-Rad). Western blots were repeated two to three times per sample. Protein abundance was quantified by densitometry (Bio-Rad GS670 Imaging densitometer).

Statistical analysis. All data are expressed as means ± SE. Data were collected from right ventricles and single right ventricular myocytes isolated from three to eight hearts per group. For RNase protection assays, the arbitrary densitometric units were normalized to the value of the cyclophilin gene. Statistical analysis was done using one-way ANOVA from the SPSS software program (version 7.0 for Windows). When ANOVA showed statistical significance by F test, intergroup comparisons were made using the Student-Newman-Keuls procedure. P < 0.05 was considered significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Effect of DITPA after MI. In our study, we analyzed rats with infarct sizes >30% (range 30-60%). Table 1 shows some of the general characteristics and hemodynamic parameters after MI and DITPA treatment. Infarct sizes were comparable among vehicle-treated post-MI and DITPA-treated post-MI animals [49.9 ± 4.1% (n = 13), 52.7 ± 3.9% (n = 4), and 53.2 ± 3.8% (n = 13) for groups treated with vehicle, 3.75 mg/kg DITPA, and 10 mg/kg DITPA, respectively]. Daily subcutaneous injections with DITPA had no effect on body weight. After 21 days of DITPA treatment, body weight increased by 5.6 ± 0.8% (n = 14) in vehicle-treated post-MI animals, by 7.1 ± 1.6% (n = 8) in 3.75 mg/kg DITPA-treated post-MI animals, and by 4.2 ± 1.5% (n = 14) in 10 mg/kg DITPA-treated post-MI animals. Lung wet weight-to-dry weight ratios were slightly increased in vehicle-treated post-MI animals compared with sham-operated animals, but this did not reach statistical significance [4.4 ± 0.2 (n = 11) vs. 3.9 ± 0.2 (n = 7); P = 0.1] and were unchanged in DITPA-treated post-MI animals [3.75 mg/kg DITPA: 3.8 ± 0.4 (n = 4); 10 mg/kg DITPA, 4.2 ± 0.2 (n = 7); P > 0.05].

                              
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  		Table 1.   General characteristics and hemodynamic parameters after myocardial infarction and DITPA treatment

The effects of MI and DITPA treatment on hemodynamic properties of the hearts are summarized in Table 1. The most notable change was in LVEDP, which was elevated after MI with or without DITPA treatment. Changes in the rate of contraction (+dP/dt) were unaffected after MI or DITPA treatment. The rate of relaxation (-dP/dt) was significantly (P < 0.05) decreased after MI and was modestly restored after DITPA treatment. The lack of significance in these parameters might be explained by the variations in infarct sizes (33).

Effect of DITPA on Ito. From this point, right ventricular myocytes derived from sham-operated animals, vehicle-treated post-MI animals, 3.75 mg/kg DITPA-treated post-MI animals, and 10 mg/kg DITPA-treated post-MI animals are referred to as sham myocytes, post-MI myocytes, 3.75 mg/kg DITPA post-MI myocytes, and 10 mg/kg DITPA post-MI myocytes, respectively. Figure 1 shows representative normalized whole cell voltage-clamp records measured from sham (Fig. 1A), post-MI (Fig. 1B), 3.75 mg/kg DITPA post-MI (Fig. 1C), and 10 mg/kg DITPA post-MI myocytes (Fig. 1D). Comparison of Fig. 1, A and B, shows that peak current density (Ipeak) was reduced in post-MI myocytes compared with that in sham myocytes. Treatment with 3.75 mg/kg DITPA modestly increased Ipeak (Fig. 1C), whereas 10 mg/kg DITPA restored Ipeak to levels observed in sham-operated controls (Fig. 1D). Ito density-voltage relationships are summarized in Fig. 1E. Ito density at +40 mV was significantly reduced from 14.0 ± 1.0 pA/pF (n = 21) in sham myocytes to 10.2 ± 0.9 pA/pF (n = 41, P = 0.001) in post-MI myocytes. To assess whether the observed changes in Ito densities resulted from alterations in the channel gating or in maximal conductance, we also calculated conductance-voltage curves (see MATERIALS AND METHODS) from the data in Fig. 1. The estimated maximal whole cell transient outward channel conductance (Gmax) was significantly reduced from 127.9 ± 9.8 pS/pF (n = 21) in sham myocytes to 84.6 ± 7.8 pS/pF (n = 41; P = 0.002) in post-MI myocytes. As summarized in Table 2, the midpoint of the conductance-voltage relationship (V1/2 act) was shifted slightly in a hyperpolarizing direction in post-MI myocytes compared with that of sham myocytes without differences in the slope factors for activation (k).


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  		Fig. 1.   Effect of 3,5-diiodothyropropionic acid (DITPA) on transient outward (Ito) and sustained currents (Isus) in right ventricular myocytes after myocardial infarction (post-MI). Normalized traces of Ito and Isus elicited by 500-ms voltage steps from -40 to +60 mV (in 20-mV steps) from a holding potential of -80 mV were derived from sham (A), post-MI (B), 3.75 mg/kg DITPA post-MI (C), and 10 mg/kg DITPA post-MI myocytes (D). Horizontal bars indicate 0 pA/pF. Current-voltage (I-V) plots are shown for Ito (E) and Isus (F) for sham, post-MI, 3.75 mg/kg DITPA post-MI, and 10 mg/kg DITPA post-MI myocytes. Myocytes were depolarized every 10 s. * P < 0.05, sham vs. post-MI myocytes; Dagger  P < 0.05, post-MI vs. 10 mg/kg DITPA post-MI myocytes.


                              
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  		Table 2.   Characteristics of the transient outward current after myocardial infarction and DITPA treatment

Treatment of post-MI animals with 3.75 mg/kg DITPA increased Ito density to 11.2 ± 0.8 pA/pF (+40 mV, n = 45; P = 0.56), whereas 10 mg/kg DITPA treatment produced a marked increase in Ito density to 15.2 ± 1.1 pA/pF (+40 mV, n = 45; P = 0.001) compared with post-MI myocytes. Changes in Gmax mirrored changes in Ito density. Compared with vehicle-treated post-MI animals, treatment with 3.75 mg/kg DITPA increased Gmax to 107.2 ± 8.3 pS/pF (n = 45) without reaching statistical significance (P > 0.05), whereas 10 mg/kg DITPA significantly increased Gmax to 133.0 ± 10.7 pS/pF (n = 45). Despite the changes in Gmax, DITPA treatment had no effect on V1/2 act or k compared with vehicle treatment. Steady-state inactivation properties of Ito were not affected after MI or DITPA treatment, as summarized in Table 2.

Effect of DITPA on Kv4.2, Kv4.3, and Kv1.4 expression. To investigate the molecular basis for the effects of DITPA on Ito, we employed RNase protection assays to measure mRNA levels of the "transient-outward like" K+ channel genes Kv1.4, Kv4.2, and Kv4.3 (10, 11). Because Ito was only marginally affected by 3.75 mg/kg DITPA, we only examined the effects of 10 mg/kg DITPA. To confirm that 10 mg/kg DITPA had the expected effects on gene expression under our experimental conditions, we examined the mRNA levels of alpha -MHC and beta -MHC because these genes are strongly regulated by hypertrophy and thyroid hormone (19). As shown in Fig. 2A, the ratio of beta -MHC to alpha -MHC increased significantly after MI [sham: 0.48 ± 0.01 (n = 3) vs. post-MI: 1.79 ± 0.07 (n = 3); P < 0.01]. Consistent with previous results (19), DITPA treatment reduced the ratio of beta -MHC to alpha -MHC [0.59 ± 0.02 (n = 3)] to levels similar to those measured in sham-operated hearts (P > 0.05). Figure 2 also shows the results of RNase protection assays for cyclophilin as well as Kv4.2 (Fig. 2B), Kv4.3 (Fig. 2C), and Kv1.4 (Fig. 2D) mRNA levels in right ventricle. Mean Kv4.2-to-cyclophilin mRNA ratios were reduced from 0.73 ± 0.03 (n = 3) in sham samples to 0.51 ± 0.02 (n = 3) in post-MI samples (P < 0.01). After DITPA treatment, Kv4.2 mRNA levels were significantly (P < 0.01) greater than those in untreated hearts [0.63 ± 0.01 (n = 3)], indicating that DITPA partially restored Kv4.2 mRNA levels. Kv4.3-to-cyclophilin mRNA ratios were also significantly decreased in vehicle-treated post-MI hearts [1.00 ± 0.06 (n = 3)] compared with sham samples [1.19 ± 0.05 (n = 3); P < 0.05], but DITPA treatment did not reverse these changes [0.889 ± 0.002 (n = 3); P > 0.05]. As shown in Fig. 2D, Kv1.4 mRNA ratios were significantly (P < 0.01) increased in post-MI samples [0.55 ± 0.03 (n = 3)] compared with sham samples [0.36 ± 0.04 (n = 3)]. DITPA treatment normalized Kv1.4 mRNA ratios to levels observed in sham-operated hearts [0.37 ± 0.02 (n = 3); P > 0.05].


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  		Fig. 2.   Effect of DITPA on genes encoding Ito and myosin heavy chain (MHC) in right ventricle. Left: representative comparison of mRNA levels are from sham (S1, S2, S3), post-MI (V1, V2, V3), and 10 mg/kg DITPA post-MI hearts (D1, D2, D3). Expression of alpha - (top) and beta -MHC (bottom) isoforms is shown in A. Expression of Kv (top) and cyclophilin-protected mRNA fragments (bottom) is shown in B-D. Right: mean changes in Kv mRNA and beta -MHC-to-alpha -MHC ratio normalized to cyclophilin mRNA levels in sham (open bars), post-MI (shaded bars), and 10 mg/kg DITPA post-MI. hearts (filled bars), respectively. * P < 0.05, sham vs. post-MI hearts; dagger  P < 0.05, sham vs. 10 mg/kg DITPA post-MI hearts; Dagger  P < 0.05, post-MI vs. 10 mg/kg DITPA post-MI hearts.

Because the level of RNA expression may not always be indicative of protein expression levels, we also measured protein levels of Kv1.4 and Kv4.2 subunits. Kv4.3 protein was also measured but was not reported because of rapid degradation of this protein under our experimental conditions. Figure 3A shows a representative Western blot of protein from adult rat brain (10 µg) and right ventricles probed with anti-Kv4.2 antibody. The antibody labeled a single band in all samples, with an approximate molecular mass of 77 kDa. As summarized in Fig. 3B, the level of Kv4.2 protein was markedly reduced (P = 0.03) in the post-MI hearts compared with that in sham-operated hearts [sham: 0.53 ± 0.09 arbitrary units (n = 3) vs. post-MI: 0.21 ± 0.04 arbitrary units (n = 3)]. Figure 3B also shows that DITPA treatment of post-MI animals restores Kv4.2 protein [0.50 ± 0.04 arbitrary units (n = 3)] to levels observed in sham-operated animals, correlating well with electrophysiological and Kv4.2 mRNA studies. Figure 3C depicts a Western blot of the same protein samples as shown in Fig. 3A, probed with anti-Kv1.4 antibody, and shows two distinct bands, in both brain and myocyte protein, as shown previously in adult and cultured neonatal rat ventricular myocytes (42, 45). Consistent with the Kv1.4 mRNA measurements, Kv1.4 immunoreactivity increased significantly from 0.43 ± 0.04 arbitrary units (P < 0.05) in sham-operated animals (n = 3) to 0.68 ± 0.15 arbitrary units in post-MI animals (n = 3). After DITPA treatment, Kv1.4 protein levels were significantly reduced below levels observed in sham-operated samples [0.224 ± 0.007 arbitrary units (n = 3); P < 0.001].


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  		Fig. 3.   Effect of DITPA on Kv channel subunit immunoreactive proteins encoding Ito in right ventricle after MI. Blots show levels of Kv4.2 (A) and Kv1.4 proteins (C) from brain (Br), sham (S1, S2, S3), post-MI (V1, V2, V3), and 10 mg/kg DITPA post-MI hearts (D1, D2, D3). Bar graphs show mean changes in Kv4.2 (B) and Kv1.4 (D) in sham (open bars), post-MI (shaded bars), and 10 mg/kg DITPA post-MI hearts (filled bars). * P < 0.05, sham vs. post-MI hearts; dagger P < 0.05, sham vs. 10 mg/kg DITPA post-MI hearts; Dagger  P < 0.05, post-MI vs. 10 mg/kg DITPA post-MI hearts.

Effect of DITPA on other ion currents. Given the effect of DITPA on Ito, we wanted to examine whether this agent could exert similar effects on other ionic currents. Figure 1F shows the current-voltage relationships for Isus evaluated at the end of a 500-ms depolarizing voltage step. At +40 mV, Isus density was not significantly different in post-MI myocytes [5.6 ± 0.3 pA/pF (n = 41)] from that in sham myocytes [6.4 ± 0.4 pA/pF (n = 21); P = 0.07]. Treatment with 3.75 mg/kg DITPA [6.0 ± 0.3 pA/pF (n = 45); P = 0.06] and 10 mg/kg DITPA [7.0 ± 0.4 pA/pF (n = 45); P = 0.06] produced no changes in Isus density compared with vehicle-treated controls. Figure 4 shows representative normalized IK1 currents recorded from sham (Fig. 4A), post-MI (Fig. 4B), 3.75 mg/kg DITPA post-MI (Fig. 4C), and 10 mg/kg DITPA post-MI myocytes (Fig. 4D). Comparison of these figures demonstrates that IK1 was lower in all post-MI myocytes than in sham myocytes. IK1 density evaluated at -130 mV was decreased significantly (P = 0.006) in post-MI myocytes [-12.1 ± 0.8 pA/pF (n = 25)] compared with that in sham myocytes [-15.7 ± 0.9 pA/pF (n = 25)]. Treatment with DITPA did not restore (P > 0.05) IK1 density (Fig. 4E) to that observed in sham myocytes [-11.3 ± 1.0 pA/pF (n = 14) for 3.75 mg/kg DITPA and -10.8 ± 1.0 pA/pF (n = 10) for 10 mg/kg DITPA evaluated at -130 mV]. Figure 5 shows representative normalized Cd2+-subtracted ICa,L traces recorded in sham (Fig. 5A), post-MI (Fig. 5B), and 10 mg/kg DITPA post-MI (Fig. 5C) myocytes, with the current-voltage relationship depicted in Fig. 5D. ICa,L density was unchanged after MI [ICa,L densities evaluated at 0 mV were -6.1 ± 0.5 pA/pF (n = 17) and -5.4 ± 0.3 pA/pF (n = 26) in myocytes from sham and post-MI myocytes, respectively; P > 0.05]. DITPA treatment did not change ICa,L compared with vehicle-treated controls [ICa,L density evaluated at 0 mV was -6.6 ± 0.4 pA/pF (n = 20); P > 0.05]. INa density and the midpoints for steady-state inactivation (V1/2 inact) were also unchanged (P > 0.05) after MI and 10 mg/kg DITPA treatment. INa density at -35 mV was -17.6 ± 1.1 (n = 21), -16.5 ± 1.5 (n = 15), and -15.0 ± 1.0 pA/pF (n = 11), and V1/2 inact was -94.7 ± 3.2 (n = 4), -93.9 ± 1.1 (n = 4), and -91.8 ± 4.8 mV (n = 3), in sham, post-MI, and 10 mg/kg DITPA post-MI myocytes, respectively.


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  		Fig. 4.   Effect of DITPA on inward (IK1) rectifier current in right ventricular myocytes after MI. Normalized IK1 traces were derived from sham (A), post-MI (B), 3.75 mg/kg DITPA post-MI (C), and 10 mg/kg DITPA post-MI myocytes (D). IK1 traces were elicited by 500-ms voltage steps from -130 to -60 mV from a holding potential of -80 mV. Horizontal bars indicate 0 pA/pF. E: IK1 were normalized to membrane capacitance and plotted against voltage for sham, post-MI, 3.75 mg/kg DITPA post-MI, and 10 mg/kg DITPA post-MI myocytes. Steady-state current measured at end of test pulse in presence of Ba2+ (0.3 mM) was subtracted from current evoked at same voltage step in absence of Ba2+. Myocytes were depolarized every 10 s. * P < 0.05, sham vs. post-MI myocytes.



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  		Fig. 5.   Effect of DITPA on L-type Ca2+ current (ICa,L) in right ventricular myocytes after MI. Representative normalized ICa,L were derived from sham (A), post-MI (B), and 10 mg/kg DITPA post-MI myocytes (C). Current traces show Cd2+-sensitive difference currents (0.3 mM CdCl2) elicited by 500-ms voltage steps to -40, 0,+10, and +30 mV from a holding potential of -80 mV. Horizontal bars indicate 0 pA/pF. D: ICa,L density was plotted against voltage for sham, post-MI, and 10 mg/kg DITPA post-MI myocytes. Myocytes were depolarized every 5 s.

Effect of DITPA on action potential. Because DITPA treatment was able to normalize Ito density without affecting other current densities, we examined whether the increase in Ito density in post-MI myocytes was associated with action potential shortening. As shown in Fig. 6, action potentials in sham myocytes typically had a spikelike appearance, whereas the rate of repolarization in post-MI myocytes was slowed, resulting in a significant prolongation of action potential duration (APD) at both the 50% (APD50) and 90% (APD90) repolarization level [APD50 was 5.0 ± 0.8 (n = 21) and 12.6 ± 1.3 ms (n = 15), P < 0.05, whereas APD90 was 30.8 ± 3.5 (n = 21) and 55.7 ± 5.7 ms (n = 15), P < 0.05, for sham and post-MI myocytes, respectively]. Both 3.75 and 10 mg/kg DITPA treatment accelerated repolarization and shortened APD compared with vehicle-treated controls. The effect of 3.75 mg/kg DITPA was most apparent for APD50 [8.2 ± 0.7 ms (n = 16); P < 0.01], whereas 10 mg/kg DITPA significantly (P < 0.01) shortened both the APD50 [8.0 ± 0.6 ms (n = 18)] and APD90 [35.3 ± 3.2 ms (n = 16] compared with vehicle-treated controls. Resting membrane potentials in post-MI myocytes were significantly (P < 0.001) depolarized [-71.1 ± 1.0 mV (n = 18)] compared with sham myocytes [-76.0 ± 1.3 mV (n = 20)]. DITPA treatment had no effect on the resting membrane potential [-69.2 ± 1.2 mV (n = 16) for 3.75 mg/kg DITPA post-MI and -71.1 ± 1.0 mV (n = 18) for 10 mg/kg DITPA post-MI; P > 0.05].


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  		Fig. 6.   Effect of DITPA on action potentials in right ventricular myocytes after MI. Representative action potential traces were recorded from sham (A), post-MI (B), 3.75 mg/kg DITPA post-MI (C), and 10 mg/kg DITPA post-MI myocytes (D). Horizontal bars indicate 0 mV. Bar graphs show mean changes in action potential duration at 50% (APD50; E) and 90% repolarization (APD90; F) in sham, post-MI, 3.75 mg/kg DITPA post-MI, and 10 mg/kg DITPA post-MI myocytes. Action potentials were elicited by a brief (5 ms) suprathreshold injection of depolarizing current (2× threshold) applied at 0.1 Hz. * P < 0.05, sham vs. post-MI myocytes; dagger  P < 0.05, sham vs. 10 mg/kg DITPA post-MI myocytes; Dagger  P < 0.05, post-MI vs. 10 mg/kg DITPA post-MI myocytes; * P < 0.05, sham vs. 3.75 mg/kg DITPA post-MI myocytes.

It is conceivable that the changes in APD after DITPA treatment could result from changes in a number of currents during an action potential. However, as shown above, the changes in APD with DITPA treatment are not due to alterations in Isus, IK1, ICa,L, or INa. The APD measurements (Fig. 6) were performed under conditions in which [Ca2+]i transients might not be entirely eliminated when 5 mM EGTA was included in the pipette. Therefore, the changes in APD with DITPA could conceivably have resulted from differences in Na+/Ca2+ exchange currents (INa/Ca) secondary to alterations in [Ca2+]i transients. Therefore, we also measured action potentials in the presence of 30 mM BAPTA in the pipette and 0.1 mM external Ca2+ to eliminate [Ca2+]i transients (R. Kaprielian and P. H. Backx, unpublished observations). Under these conditions, the APD50 [4.4 ± 0.8 (n = 6) and 10.5 ± 3.6 ms (n = 5) for sham and post-MI myocytes, respectively; P < 0.01] and APD90 (27.7 ± 4.6 and 49.1 ± 7.8 ms for sham and post-MI myocytes, respectively; P < 0.01) were prolonged after MI. DITPA treatment (at 10 mg/kg) restored APD50 [4.9 ± 0.7 ms (n = 6)] and APD90 [31.2 ± 3.5 ms (n = 6)] to levels observed in sham myocytes.

To determine whether the changes in APD recorded in single cells under relatively nonphysiological conditions might be predictive of changes occurring in the whole heart, we recorded monophasic action potentials from the right ventricular wall in Langendorff-perfused hearts at 37°C. Typical examples of monophasic action potentials are shown in Fig. 7. Consistent with the single-cell electrophysiology, monophasic APD50 [17.9 ± 1.2 (n = 13) and 24.8 ± 1.9 ms (n = 9); P < 0.01] and APD90 [42.9 ± 2.1 (n = 13) and 52.5 ± 5.4 ms (n = 9); P < 0.01] were significantly prolonged after MI. DITPA treatment (at 10 mg/kg) normalized the monophasic APD in hearts from post-MI animals to values similar to those observed in hearts from sham-operated animals [monophasic APD50: 14.2 ± 0.8 ms (n = 9); monophasic APD90: 37.6 ± 1.1 ms (n = 9)].


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  		Fig. 7.   Effect of DITPA on monophasic action potentials in right ventricular free wall after MI. Recordings were obtained from sham (A), post-MI (B), and 10 mg/kg DITPA post-MI hearts (C). Bar graphs show mean changes in APD50 (D) and APD90 (E) in sham, post-MI, and 10 mg/kg DITPA post-MI hearts. * P < 0.05, sham vs. post-MI hearts; dagger  P < 0.05, sham vs. 10 mg/kg DITPA post-MI hearts; Dagger  P < 0.05, post-MI vs. 10 mg/kg DITPA post-MI hearts.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Most previous studies of heart disease following left ventricular infarction have reported cellular, electrical, and mechanical changes in the right and left ventricles (1, 3, 21, 24, 30, 35, 37). In our studies we examined the changes in Ito and APDs that occur in the right ventricle after MI of the left ventricle. The right ventricle was chosen for these studies because changes in the right ventricle are important determinants of the prognosis in heart disease (49) and isolation of myocytes from the right ventricle avoids potential problems associated the presence of a large scar in the infarcted left ventricle. In addition, hypertrophic remodeling in the right ventricle exceeds that occurring in the noninfarcted left ventricle (data not shown). Alterations in the right ventricle following loss of left ventricular myocytes as a result of infarction are not unexpected for a number of reasons. For example, reductions in left ventricular contractility will directly increase the load on the right ventricle. Left ventricular changes can also affect right ventricular properties as a consequence of direct mechanical coupling between the two ventricular chambers. Furthermore, neurohumoral activation following infarction will produce circulating local factors that may affect both ventricles (1, 36, 37).

Effect of DITPA on Ito. Downregulation of Ito is consistently observed in human patients (27, 28) and in a variety of heart disease models (20, 21, 24, 35). Consistent with previous observations (21, 35), Ito density was reduced in myocytes from post-MI hearts, but this reduction only reached statistical significance at voltages above +30 mV. Our inability to detect significant changes in Ito density at potentials below +30 mV is unclear but might result from the relatively small current magnitude at these voltages coupled with biological and experimental variability of the whole cell current measurements in myocytes. Alternatively, the lack of significant reduction in Ito at less positive potentials might originate from the positive shift (about +5 mV) in the V1/2 of Ito activation observed in the post-MI group.

Gmax and Ito were significantly reduced in post-MI myocytes. This decrease in Gmax could result from either reductions in the number of channels or changes in the maximum probability of channel opening, or both. However, the reductions in Kv4.2 and Kv4.3 expression were very similar to those for Gmax, suggesting that reductions in channel density were primarily responsible for the decline in Ito density and Gmax. These results are consistent with previous studies showing that Kv4.2 and Kv4.3 are the major contributors to Ito in the adult rodent (11, 15, 21, 39, 47). In contrast, the expression of the "fetal" K+ channel gene Kv1.4 was increased after MI, as reported previously (21, 45, 47), and similar to that observed in hypertensive rats (26). The significance of increases in Kv1.4 expression remains unclear, but it is interesting to note that the directional shifts in V1/2 matched those expected from shifts in the expression of Kv4.2-based currents to Kv1.4-based currents (46).

Because thyroid hormone can increase the expression of genes encoding for Ito (17, 39, 45) and alter the biophysical properties of Ito (38, 39, 45), we were interested in examining whether agents with thyroid hormone-like activity could reverse some of the right ventricular electrical remodeling that occurred in our model. The effects of thyroid hormone itself, however, may be complicated by noncardiac effects (14). We chose, instead, to examine the effects of the thyroid hormone analog DITPA, because this agent reportedly exerts only minor effects on heart rate and metabolism compared with thyroid hormone itself (25, 32) and might, therefore, have clinical value. DITPA treatment restored Ito density and Gmax in post-MI myocytes to levels observed in sham cells but did not affect the V1/2 of activation. These increases in Gmax after DITPA treatment were accompanied by elevations in Kv4.2 expression and decreases in Kv1.4 expression, reversing the pattern of changes observed after MI. The effects of DITPA on K+ channel expression appear to be mediated at the transcriptional level, consistent with the known effects of thyroid hormone on Kv4.2 and Kv1.4 transcription in cardiac myocytes (17, 29, 39, 45). In contrast, Kv4.3 RNA levels were unchanged after DITPA treatment, consistent with previous studies using thyroid hormones in adult rats (29, 39). These differential effects of DITPA treatment on Kv4.2, Kv4.3, and Kv1.4 might explain the inability of this drug to reverse the shift in V1/2 of activation observed in post-MI myocytes.

Effect of DITPA on APD and membrane potentials. Consistent with previous observations, we observed action potential prolongation in noninfarcted right ventricular myocytes after MI (21, 35). Our study further shows that DITPA treatment in post-MI hearts accelerated action potential repolarization back to control values. The modulation of APD in isolated myocytes by MI and DITPA treatment coincided closely with changes in Ito, suggesting that the alterations in Ito density were responsible for the differences in APD among the groups. It should be recognized, however, that the divalent cation Cd2+ was present when Ito was recorded but was absent when action potentials were recorded. This could help explain the observation that Ito densities were only significantly reduced at the more positive potentials in the post-MI group despite significant reductions in Gmax, because Cd2+ potently shifts the V1/2 for Ito activation to more depolarized voltages (2, 46).

The observed changes in APD among the different groups could also conceivably involve alterations in a number of depolarizing and/or hyperpolarizing currents. Our studies establish that Isus, ICa,L, or INa densities and their gating properties were not affected by MI or DITPA treatment. However, changes in other currents besides Ito might contribute to the observed alterations in action potential profile among the groups. Previous studies reported that INa/Ca were either increased (24) or decreased (50) after MI and that the exchanger activity was either decreased (5) or unchanged (6) after daily thyroid hormone treatment. In some of our studies any contribution of INa/Ca to the action potential were minimized either by including 5 mM EGTA in the pipette or by reducing extracellular Ca2+ to 0.1 mM and including 30 mM BAPTA in the pipette. Therefore, the changes in APD under these experimental conditions, at least in the single-cell recordings, are unlikely to result from differences in INa/Ca among the groups.

Monophasic action potential recordings from whole hearts showed similar APD differences among groups to those observed in isolated myocyte experiments, suggesting that the isolated myocyte studies are representative of global changes in the intact heart. However, the isolated myocyte studies were performed under relatively nonphysiological conditions (with respect to temperature, frequency, and cellular dialysis) compared with the isolated heart measurements, making it possible that one or more other mechanisms might also contribute to the pattern of changes in APD. Extrapolation, therefore, from isolated cells to the whole heart should be done with caution.

IK1 was significantly reduced at hyperpolarized potentials but not at depolarized potentials (positive to -90 mV) after MI. As mentioned previously for Ito, the lack of detectable changes in IK1 at depolarized potentials might result from the relatively small magnitude (34) of the current at these potentials coupled with the inherent biological and experimental variability in these whole cell voltage-clamp recordings. Alternatively, MI could have conceivably altered the properties of IK1 differently as a function of membrane potential. Regardless, depolarization of resting membrane potentials after MI is consistent with decreased IK1 densities, although changes in other background currents (i.e., Cl- and ATP-sensitive K+ currents) might also contribute to these alterations. Nevertheless, the inability of DITPA treatment to reverse changes in either IK1 or resting membrane potential are consistent with a connection between the alterations of these parameters after MI.

Limitations of the present study. Our studies show that APD in myocytes was prolonged after MI. DITPA treatment shortened the duration of myocytes from infarcted hearts back to control levels. The APD in the different groups coincided with Ito density but not with INa, ICa,L, Isus, or IK1 density, suggesting that changes in Ito density are responsible for the observed alterations in APD. However, it is certainly possible that other currents, not measured in this study, may also change after infarction and DITPA treatment and thereby contribute to the observed changes in APD. Because the primary aim of our study was to determine the effects of infarction and DITPA on Ito, the investigation of these other currents is beyond the scope of the present study.

Similar changes in APD after MI and DITPA treatment were seen in both single-myocyte studies and in whole heart monophasic action potential recordings. These data tend to suggest that the findings of the single-myocyte studies, under rather nonphysiological conditions, are predictive of global properties of the heart under more physiological conditions. Nevertheless, it is clearly possible that the mechanism(s) underlying the changes in APD in these two conditions is different.

The duration of the cardiac action potential is not uniform throughout the mammalian heart. Regional heterogeneity of APD exists in the normal myocardium (9, 23, 28) and is modulated by hypertrophy (16). Nonuniform alterations in repolarization within the myocardium may create conditions favorable for the initiation and/or propagation of reentrant arrhythmias. In the present study we made no attempt to determine whether the effects of MI and DITPA treatment varied among different regions of the heart. We also did not investigate transmural variations of APD within the right ventricular free wall because of technical difficulties associated with separation of the epicardial and endocardial layers. The question as to how MI and DITPA treatment affect transmural heterogeneity in the right ventricular free wall may be best studied in a larger species.

In conclusion, we show that DITPA treatment in post-MI rats can restore Ito density and increase Kv4.2 expression to levels observed in sham-operated controls. DITPA treatment did not affect Isus, IK1, ICa,L, or INa. Consistent with the increase in expression of Ito, a shortening of the APD was observed after DITPA treatment. These data suggest that thyroid hormones and analogs might be useful in the reversal of electrical remodeling observed after MI.


    ACKNOWLEDGEMENTS

A. D. Wickenden and R. Kaprielian contributed equally to this work.


    FOOTNOTES

We thank Dr. Fayez Dawood for performing the surgeries. We also extend special thanks to Tin Nguyen for performing RNase protection and Western blot assays. We are grateful to the Tiffen Trust Fund and the Center for Cardiovascular Research at the University of Toronto for equipment grants.

This study was supported by a grant from the Heart and Stroke Foundation of Ontario (HSFO). P. H. Backx received a Medical Research Council (MRC) scholarship and is a career investigator of the HSFO. R. Kaprielian holds an MRC Doctoral Research award.

Present address of A. D. Wickenden: ICAgen Inc., 4222 Emperor Blvd., Ste. 460, Durham, NC 27703.

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: P. H. Backx, Univ. Health Network, Center for Cardiovascular Research, 101 College St., CCRW 3-802, Toronto, Ontario, Canada M5G 2C4 (E-mail: p.backx{at}utoronto.ca).

Received 25 January 1999; accepted in final form 21 September 1999.


    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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