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Relationship between K⁺ channel down-regulation and [Ca²⁺]_i in rat ventricular myocytes following myocardial infarction

R. Kaprielian, A. D. Wickenden, Z. Kassiri, T. G. Parker, P. P. Liu and P. H. Backx *

MS 8708 Received 8 September 1998; accepted after revision 27 January 1999.

	ABSTRACT

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1. Cardiac hypertrophy and prolongation of the cardiac action potential are hallmark features of heart disease. We examined the molecular mechanisms and the functional consequences of this action potential prolongation on calcium handling in right ventricular myocytes obtained from rats 8 weeks following ligation of the left anterior descending coronary artery (post-myocardial infarction (MI) myocytes).

2. Compared with myocytes from sham-operated rats (sham myocytes), post-MI myocytes showed significant reductions in transient outward K⁺ current (I_to) density (sham 19·7 ± 1·1 pA pF^-1 versus post-MI 11·0 ± 1·3 pA pF^-1; means ± s.e.m.), inward rectifier K⁺ current density (sham -13·7 ± 0·6 pA pF^-1 versus post-MI -10·3 ± 0·9 pA pF^-1) and resting membrane potential (sham -84·4 ± 1·3 mV versus post-MI -74·1 ± 2·6 mV). Depressed I_to amplitude correlated with significant reductions in Kv4.2 and Kv4.3 mRNA and Kv4.2 protein levels. Kv1.4 mRNA and protein levels were increased and coincided with the appearance of a slow component of recovery from inactivation for I_to.

3. In current-clamp recordings, post-MI myocytes showed a significant increase in [Ca²⁺]_i transient amplitude compared with sham myocytes. Using voltage-clamp depolarizations, no intrinsic differences in Ca²⁺ handling by the sarcoplasmic reticulum or in L-type Ca²⁺ channel density (I_Ca,L) were detected between the groups.

4. Stimulation of post-MI myocytes with an action potential derived from a sham myocyte reduced the [Ca²⁺] transient amplitude to the sham level and vice versa.

5. The net Ca²⁺ influx per beat via I_Ca,L was increased about 2-fold in myocytes stimulated with post-MI action potentials compared with sham action potentials.

6. Our findings demonstrate that reductions in K⁺ channel expression in post-MI myocytes prolong action potential duration resulting in elevated Ca²⁺ influx and [Ca²⁺]_i transients.

	INTRODUCTION

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Ischaemic heart disease as a result of myocardial infarction (MI) is the most prominent aetiology in the majority of patients who develop congestive heart failure (Teerlink et al. 1991). The loss of myocytes following MI causes a decrease in cardiac output which in turn leads to activation of neurohumoral systems that act initially to compensate for the loss of myocardial mass, but which in the long term can become cardiomyotoxic leading to the progression of the disease (Rouleau, 1996). During the period of compensation, hearts generally undergo hypertrophy with significant cellular and molecular remodelling of both the left and right ventricles resulting in functional and biochemical alterations of the myocardium (Beuckelmann et al. 1991, 1992, 1993, 1995; Bailly et al. 1997).

One common animal paradigm used to study MI is the rat coronary artery ligation model. It has been shown that left ventricular myocardial infarction in rats is associated with elevated right ventricular systolic pressure and hypertrophy of the free wall of the right ventricle (Pfeffer et al. 1979; Capasso et al. 1993; De Tombe et al. 1996). Most previous studies in heart disease following MI have focused on remodelling changes in the left ventricle (Orenstein et al. 1995; Anand et al. 1997), but changes in the right ventricle are also important determinants in the prognosis of heart disease (Yu et al. 1996). In this regard, considerable remodelling and hypertrophy also occurs in the right ventricle (Sethi et al. 1997) with changes in the contractile properties (De Tombe et al. 1996), [Ca²⁺]_i transients (Cheung et al. 1994; Zhang et al. 1995) and sarcoplasmic reticulum (SR) Ca²⁺-ATPase reported (Afzal & Dhalla, 1992). Alterations in the right ventricle following left ventricular infarction are not unexpected since neurohumoral activation produces circulating factors that will affect both ventricles (Afzal & Dhalla, 1992; Sethi et al. 1997). Furthermore, impairment of left ventricular output will affect right ventricular load by increasing its afterload via elevations in pulmonary arterial pressure (Pfeffer et al. 1979).

Prolongation of action potential duration has also been reported in rat left ventricular myocardium following infarction (Qin et al. 1996; Rozanski et al. 1998), as observed in patients with cardiomyopathies (Bailly et al. 1997) and terminal heart failure (Beuckelmann et al. 1993). The cause of action potential prolongation has been correlated with a decrease in repolarizing K⁺ currents in cells from failing human hearts (Beuckelmann et al. 1993) and animal models of heart disease (Qin et al. 1996; Rozanski et al. 1998). While the precise electrical alterations are likely to vary with the stage of the disease (Cerbai et al. 1994; Rozanski et al. 1998), it has been suggested that action potential prolongation can alter electrical heterogeneity (Gomez et al. 1997) and promote various types of arrhythmias leading to sudden death (Tomaselli et al. 1994). Another possible consequence of action potential prolongation is increased [Ca²⁺]_i transient amplitude (Brooksby et al. 1993; Bouchard et al. 1995; Wickenden et al. 1998) which, while expected to offset the reduced eject properties of the compromised infarcted heart, can lead to delayed after-depolarization arrhythmias (Tomaselli et al. 1994). With the level of expression of various K⁺ channels (Brahmajothi et al. 1996) and the magnitude of K⁺ currents (Clark et al. 1993) varying regionally, it is conceivable that distinct changes in right and left ventricles and Ca²⁺ handling properties also occur.

Accordingly, the aim of the present study was to examine the electrical changes in rat right ventricular myocytes derived from hearts 8 weeks following infarction. Furthermore, our studies were designed to identify the molecular basis for these changes and to elucidate the consequences of these changes on [Ca²⁺]_i. A preliminary account of some of the findings has appeared (Kaprielian et al. 1996).

	METHODS

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Left anterior descending coronary artery ligation procedure

Left anterior descending coronary artery ligations were performed on male LBN-F1 rats (Harlan, Indianapolis, IN, USA) at 10-12 weeks of age using the methods outlined previously (Orenstein et al. 1995). Animals were anaesthetized with ketamine hydrochloride (45 mg kg^-1) and xylazine (5 mg kg^-1) intraperitoneally. Once anaesthethized, rats were intubated with a 14 gauge polyethylene catheter and ventilated with room air using a small animal ventilator (model 683; Harvard Apparatus, Boston, MA, USA). Under aseptic conditions, a left thoracotomy was performed in the fifth intercostal space and the pericardium opened. The proximal left coronary artery was encircled and ligated using a 6-0 silk suture. The muscle and skin were closed in layers. Sham-operated animals were treated identically except the left coronary artery was not tied. All experimental protocols were approved by the Committee on Animal Research at the Toronto General Hospital and were performed in accordance with the 'Position of the American Heart Association on Research Animal Use'. After the surgical procedure, rats were given antibiotics, housed in a climate-controlled environment at an ambient temperature of 21°C with a 12 h light-12 h dark cycle. Water and standard Purina rat chow were given ad libitum.

Eight weeks after surgery all rats were given anti-coagulant (500 units heparin I.P.) and anaesthetized (60 mg kg^-1 pentobarbitone sodium I.P.). Once an adequate depth of anaesthesia was reached the hearts were quickly removed.

Isolation of right ventricular myocytes

Hearts were cannulated as previously described (Wickenden et al. 1997) and retrogradely perfused via the aorta for about 3 min with a calcium-containing standard Tyrode solution of the following composition (mM): 140 NaCl, 5·4 KCl, 10 Hepes, 1 MgCl₂, 1 CaCl₂, 10 D-glucose, adjusted to pH 7·4 with NaOH at 37°C. The heart was then perfused with Ca²⁺-free standard Tyrode solution for 5 min before the hearts were digested in the same solution containing collagenase (Type II, 0·55 mg ml^-1, Boehringer-Mannheim) and protease (Type XIV, 0·05 mg ml^-1, Sigma) for 8-9 min. The enzyme solution was subsequently removed by perfusing with a Kraft-Brühe (high K⁺) solution (containing (mM): 120 potassium glutamate, 20 KCl, 20 Hepes, 1 MgCl₂, 0·3 K-EGTA, 10 D-glucose) for 5 min. Following the enzyme washout period, the atria and blood vessels were removed and the ventricles were separated. All solutions were pre-bubbled with 100 % O₂ for 5 min. 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 bovine serum albumin (0·02 % w/v). Myocytes were then filtered through a nylon mesh and re-suspended in Kraft-Brühe solution containing 50 mg ml^-1 gentamicin. Calcium-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^-1.

Electrophysiological measurements in right ventricular myocytes

Current densities and action potentials were recorded using the whole-cell patch-clamp technique (Hamill et al. 1981) with an Axopatch 200A amplifier (Axon Instruments). Microelectrodes were pulled from thin-walled borosilicate glass (1·5 mm diameter; World Precision Instruments) using a Flaming-Brown micropipette puller (Sutter Instruments). The pipette tip was heat polished with a heating filament. When filled with intracellular solutions, tip resistances were typically 2-4 M. Series resistance compensation ranged between 60 and 90 %. After membrane rupture, the cell capacitance was estimated by integrating the area of the capacitance transients following a 5 mV step from a holding potential of -70 mV. All experiments were performed at room temperature (19-21°C). Currents were digitized at 2 kHz and stored off-line for analysis.

In order to measure L-type Ca²⁺ currents (I_Ca,L), voltage-clamp recordings were made in myocytes superfused with a solution containing (mM): 140 NaCl, 1 MgCl₂, 10 Hepes, 4 CsCl, 1 CaCl₂, 10 D-glucose, adjusted to pH 7·4 with NaOH. I_Ca,L was also recorded using the action potential voltage-clamp technique in myocytes superfused with a solution containing (mM): 145 CsCl, 10 Hepes, 1 MgCl₂, 2 CaCl₂, 10 D-glucose, adjusted to pH 7·4 with CsOH. Intracellular solutions always contained (mM): 150 CsCl, 10 Hepes, 1 MgCl₂, 5 EGTA, 5 MgATP, adjusted to pH 7·2 with CsOH. Na⁺- and K⁺-free extracellular solutions were used during action potential voltage-clamp recordings to minimize K⁺, Na⁺ and Na⁺-Ca²⁺ exchange currents activated during an action potential. These solutions contained (mM): 150 CsCl, 1 MgCl₂, 10 Hepes, 2 CaCl₂, 10 D-glucose, adjusted to pH 7·4 with CsOH. Sham myocytes were stimulated with short or long action potential waveforms which were derived from sham or post-MI hearts, respectively. Due to the heterogeneous nature of action potential waveform, we recorded 20 action potentials from five hearts in right ventricular myocytes before selecting action potentials as command waveforms for each respective group. Action potential waveforms that most closely approached the mean action potential duration at 50 and 90 % repolarization were used to examine the Ca²⁺ currents under action potential voltage-clamp conditions. I_Ca,L was estimated as the current in the absence of CdCl₂ minus the current remaining after the addition of 0·3 mM CdCl₂. The difference currents were integrated to estimate the total influx of calcium (I_Ca,L).

For K⁺ currents and action potential recordings, myocytes were superfused with a standard Tyrode solution containing (mM): 140 NaCl, 10 Hepes, 1 MgCl₂, 4 KCl, 1 CaCl₂, 10 D-glucose, adjusted to pH 7·4 with NaOH. CdCl₂ (0·3 mM) was routinely added for K⁺ current recordings to block I_Ca,L. Intracellular solution for K⁺ currents and action potentials had the following composition (mM): 130 potassium aspartate, 20 KCl, 10 Hepes, 1 MgCl₂, 5 NaCl, 5 EGTA, 5 MgATP, adjusted to pH 7·2 with Trizma base. Action potentials were corrected by -9 mV to compensate for liquid junction potentials that arose from the use of potassium aspartate in microelectrodes.

Fluorescence measurements in right ventricular myocytes

Fluorescence measurements were performed using light from a 75 W xenon lamp (Oriel Corp, Stratford, CT, USA) passed through bandpass filters (Omega Optical) centred at 340 or 380 nm via an epifluorescence port and a × 40 Fluor objective lens (Nikon). The emitted fluorescence was collected by the objective lens and passed through a 510 nm filter to a photomultiplier (R2368, Hamamatsu). The photomultiplier output was filtered at 100 Hz, recorded using an A/D data acquisition board (2801A, Data Translation) and stored in the computer for analysis. The ratio of the background- subtracted fluorescence signal (340/380) was used to estimate [Ca²⁺]_i using the equation given by Grynkiewicz et al. (1985):

where K_D' is the apparent dissociation constant, R is the ratio of the background-subtracted fluorescence at 340 nm excitation to that at 380 nm excitation. The effective dissociation constant (K_D') is defined as K_D × where K_D is the dissociation constant of fura-2 for Ca²⁺ which is 0·22 µM in the presence of 1 mM MgCl₂ (Molecular Probes - Handbook of fluorescent probes and research chemicals, 1996). In our experiments , which is defined as the ratio of fluorescence measured with 380 nm excitation light in the absence of Ca²⁺ to that measured at saturating levels of [Ca²⁺]_i (10 mM), was 10·2. The fluoresence ratio in the presence of saturating Ca²⁺ (R_max) was 6·43 and that in the absence of Ca²⁺ (R_min) was 0·20. Background fluorescence was measured at both wavelengths after a gigaohm seal was obtained and prior to rupturing the cell membrane.

[Ca²⁺]_i was measured in right ventricular myocytes under current-clamp, action potential voltage-clamp and voltage-clamp conditions. For action potential voltage-clamp experiments, the action potential waveform from either sham or post-MI myocytes was used as the command signal to drive the membrane potential of the same cell and [Ca²⁺]_i was recorded. For voltage-clamp recordings, sham and post-MI myocytes short pulses (100 ms duration) were applied from a holding potential of -80 mV to +10 mV. All [Ca²⁺]_i measurements were made under steady-state conditions by stimulating the myocyte at 0·25 Hz and recording fluorescence at both wavelengths between the 17th and 20th beat. Bath solution consisted of a standard Tyrode solution containing 2 mM CaCl₂. The intracellular solution contained (mM): 130 potassium aspartate, 20 KCl, 10 Hepes, 1 MgCl₂, 6 NaCl, 0·075 fura-2 pentopotassium salt, 5 MgATP, adjusted to pH 7·2 with Trizma base.

SR calcium content in right ventricular myocytes

SR Ca²⁺ content was estimated by applying a train of 10 conditioning pulses from -70 to +10 mV (100 ms clamp steps separated by 1 s intervals) to establish uniform SR loading conditions. Bath solutions consisted of a modified Tyrode solution containing (mM): 140 NaCl, 10 Hepes, 1 MgCl₂, 4 CsCl, 2 CaCl₂, adjusted to pH 7·4 using CsOH. Recording electrodes contained (mM): 150 CsCl, 10 Hepes, 1 MgCl₂, 0·1 EGTA, 5 MgATP, adjusted to pH 7·2 with CsOH. After loading the SR, myocytes were rapidly superfused with the modified Tyrode solution containing 10 mM caffeine. The application of caffeine induced a large contraction accompanied by an inward current (I_Na-Ca) which was previously shown to be caused by the Na⁺-Ca²⁺ exchanger, which extrudes the Ca²⁺ released by the SR (Varro et al. 1993). I_Na-Ca was estimated as the difference in inward current induced by caffeine exposure in the presence and absence of sodium chloride (equimolar substitution with tetraethylammonium chloride). During caffeine applications, holding potential was set at -70 mV to enhance I_Na-Ca and the SR Ca²⁺ content was calculated by integrating I_Na-Ca as described by Varro et al. (1993). Cell volume was calculated from membrane capacitance and then converted to volume by assuming a surface to volume ratio of 0·5 µm^-1 (Page, 1978) and a specific capacity of 1 µF cm^-2.

Preparation of total RNA from the right ventricle and RNase protection assays

Hearts were removed rapidly, the right ventricle was isolated, rinsed briefly in a standard Tyrode solution and snap frozen in liquid nitrogen. Ventricular tissue was powdered and RNA extracted by the one-step acid guanidium thiocyanate-phenol-chloroform method (Chomczynski & Sacchi, 1987). The concentration of RNA was measured spectrophotometrically and confirmed by agarose gel electrophoresis. Gels were loaded with 10 µg total RNA obtained from seven hearts for each group. RNA levels were quantified by densitometry and mRNA levels were normalized to cyclophilin mRNA levels in the same sample. RNase protection assays (RPA) were performed using an RPAII ribonuclease protection assay kit (Ambion, Austin, TX, USA) according to the manufacturer's instructions. The Kvx and IRK probes for RNase protection assays were kindly provided by Dr David McKinnon (State University of New York at Stony Brook) and have been described previously (Dixon & McKinnon, 1994). The cyclophilin probe was purchased from Ambion. Antisense probes were labelled with [-³²P]-UTP (3000 Ci mmol^-1, Mandel Scientific, Guelph, Ontario, Canada) by in vitro transcription using T7 RNA polymerase. Probes (2 ng, 2 × 10⁴ c.p.m.) were added separately to 10 µg total RNA in 20 µl of 300 mM sodium acetate, pH 6·4, containing 100 mM sodium citrate, 80 mM formamide and 1 mM EDTA. Probe and RNA were hybridized for 18 h at 45°C and the unannealed RNA was digested with RNase A (5 units ml^-1) and RNase T1 (200 units ml^-1) at 37°C for 30 min. RNase resistant hybrids were recovered, analysed on 8 M urea-4 to 6 % polyacrylamide sequencing gels and visualized by autoradiography. 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 (Gidh-Jain et al. 1996).

Western blot analysis

Rat right ventricles and brains were quickly rinsed in a standard Tyrode solution and snap frozen in liquid nitrogen and stored at -70°C for Western blot analysis. Frozen tissue was homogenized in 10 volumes of 0·3 M sucrose and 30 mM histidine. This homogenate was then centrifuged at 3000 g for 15 min. The collected supernatant was re-centrifuged at 4500 g for 1 h to precipitate membrane proteins. The membrane pellet was re-suspended 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 the protein assay using the Lowry method. All solutions in this procedure were chilled on ice and contained protein inhibitors (0·1 mM PMSF, 5 µg ml^-1 of aprotinin, leupeptin, antipain and pepstatin).

For Western blot analysis, 50-100 µg total heart protein and 10-20 µg total brain protein were fractionated on a 10 % polyacrylamide-SDS gel. After electrophoretic transfer to polyvinyldifluoride (Bio-Rad), the membranes were incubated with Kv1.4 and Kv4.2 antisera (generously provided by Dr Owen T. Jones, Toronto Western Hospital, Toronto, Canada). Bound primary antibody was detected with horseradish peroxidase-conjugated goat anti-rabbit IgG (Amersham) in the blocking buffer for 1 h at room temperature. The membrane was washed again with Tris-buffered saline (TBS) containing 0·05 % Tween-20 and 1 % Triton X-100. Immunoreactivity was detected using the enhanced chemiluminescence (ECL) reagent (Amersham) and quantified by densitometry of the developed film. Quantitative analysis was performed using Molecular Analyst software (Bio-Rad).

Data analysis

All data are presented as means ± S.E.M. with the number of cells/preparations given in parentheses (n = a/b). Comparisons of all recordings in cells from sham-operated and post-MI hearts were performed using Student's two-tailed unpaired t test or two-way analysis of variance (ANOVA), whenever necessary. Steady-state activation (g) and inactivation (h ) curves were fitted to the following Boltzmann functions:

g = 1/(1 + exp((-V - V_½)/k))

and

h = 1/(1 + exp((V - V_½)/k)),

where V is the step or conditioning potential, V_½ is the mid-point of the function and k is the slope factor. Monoexponential or biexponential functions were used to fit recovery from inactivation data.

For monoexponential fits:

I/I_o = 100 - (A_fast exp(-x/_fast));

and for biexponential fits:

I/I_o = 100 - (A_fast exp(-x/_fast)) + ((100 - A_fast) × exp(-x/_slow)),

where A_fast and _fast are the amplitude and time constant for the fast component of recovery, 100 - A_fast (i.e. A_slow) and _slow represent the amplitude and time constant for the slow component and x is the time spent at the recovery potential. We calculated the ² value to determine whether biexponential fits to the data gave significantly superior fits compared with monoexponential fits. An experimental alpha level of P < 0·05 was considered statistically significant.

	RESULTS

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Following left coronary artery ligation, the normal myocardium of the left ventricular free wall was replaced to varying degrees by connective tissue. Infarct size was assessed 8 weeks after MI by dissecting the left ventricular free wall and measuring the fraction of the left ventricular free wall which was replaced with fibrous tissue. The mean infarct sizes were 51·4 ± 2·4 % (range, 39·4-64·0 %) in the epicardium and 37·9 ± 2·9 % (range, 27·6-48·9 %) in the endocardium (20 rats). Six rats in the post-MI group died shortly after surgery, while another five developed small infarctions (< 20 %) and were excluded from the study based on previously defined criteria (Pfeffer et al. 1979; Orenstein et al. 1995). Table 1 summarizes some of the observed differences between the sham-operated and post-MI hearts measured 8 weeks following surgery. Compared with sham-operated hearts, post-MI hearts showed a 2-fold increase in the right ventricle-to-body weight ratio, pulmonary congestion measured using the lung wet-to-dry weight ratio and a significant increase in membrane capacitance (215 ± 12 versus 123 ± 4 pF, P < 0·05).

Table 1. Changes associated with myocardial infarction

Membrane potential changes following infarction

Initially we sought to examine the electrical changes in right ventricular myocytes. Right ventricular myocytes derived from sham-operated hearts will be referred to as 'sham myocytes' whereas those derived from infarcted hearts will be referred to as 'post-MI myocytes'. Figure 1 shows that action potentials were significantly prolonged in post-MI myocytes compared with sham myocytes when evaluated at either 50 % repolarization (sham 4·8 ± 0·7 ms (n = 29/10) versus post-MI 13·5 ± 3·6 ms (n = 13/6), P < 0·01) or 90 % repolarization (sham 29·3 ± 3·4 ms (n = 29/10) versus post-MI 75·8 ± 16·3 ms (n = 13/6), P < 0·01). In addition to alterations in action potential profile, the resting membrane potential in post-MI myocytes was significantly (P < 0·001) depolarized by 10 mV compared with sham with no effect on the peak of the action potential (Table 1).

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Figure 1. Action potential characteristics in right ventricular myocytes following myocardial infarction A, representative action potential traces from a sham and a post-MI myocyte. Action potentials were elicited by a brief (5 ms) suprathreshold (2 × threshold) pulse applied at 0·2 Hz. Intracellular solutions contained 5 mM EGTA. Horizontal bars indicate 0 mV. B, mean action potential duration (APD) evaluated at 50 and 90 % (APD50 () and APD90 (), respectively) repolarization for sham (n = 29/10) and post-MI (n = 20/6) myocytes. *P < 0·05 for APD50 and APD90 between sham and post-MI myocytes.

Inward rectifier currents and channels

Depolarized resting membrane potential in post-MI myocytes compared with that in sham myocytes suggests possible changes in inward rectifier (I_K1) currents. Figure 2A illustrates representative barium-subtracted I_K1 traces recorded during 500 ms step depolarizations from -130 to -10 mV from a holding potential of -80 mV from a typical sham (left panel) and post-MI (right panel) myocyte. Na⁺ currents were inactivated by 70 ms prepulses to -40 mV. The steady-state current measured at the end of the test pulse in the presence of barium was subtracted from the current evoked at the same voltage step in the absence of barium and normalized for cellular capacitance. Figure 2B plots the current-voltage (I-V) relationships demonstrating that the barium-subtracted I_K1 densities were significantly reduced (P < 0·05) in post-MI myocytes compared with sham myocytes at -120 mV. At voltages above -90 mV, I_K1 was reduced in post-MI myocytes, but the reduction was not significant probably because of its small magnitude. Despite differences in I_K1 current, Fig. 2C shows that mRNA levels of IRK1 and IRK2, which are thought to encode the cardiac I_K1 (Kubo et al. 1993), were not significantly altered.

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Figure 2. IK1 in right ventricular myocytes following myocardial infarction A, normalized traces of the inward rectifier current (IK1) in a sham and a post-MI myocyte elicited by 500 ms voltage steps over the range -130 to -10 mV in +10 mV increments from a holding potential of -80 mV. CdCl2 (0·3 mM) was added to avoid contamination by the calcium current. Arrows indicate 0 pA pF-1. B, IK1 current density plotted against the test potential for sham (, n = 29/10) and post-MI (, n = 13/6) myocytes. The current measured at the end of the test pulse in the presence of barium was subtracted from the current in the absence of barium (protocol shown in inset). Myocytes were depolarized every 5 s. *P < 0·05 between sham and post-MI myocytes at -120 mV. C, mRNA levels for IRK1 (left panel) and IRK2 (right panel) in sham (left lanes) and post-MI (right lanes) right ventricles were measured using an RNase protection assay (RPA). The upper and lower bands represent IRKx- and cyclophilin-protected mRNA fragments, respectively. The bar graphs show the mean mRNA levels in post-MI () hearts expressed as a percentage of the levels observed in sham () hearts (i.e. % Sham).

Transient outward and sustained currents

The voltage-dependent transient outward current (I_to) is a major K⁺ current in rat which contributes to the rapid membrane repolarization (Apkon & Nerbonne, 1991). Figure 3A shows representative I_to traces in a sham and a post-MI myocyte following 500 ms step depolarizations to a range of voltages from -30 to +70 mV while holding at -80 mV. In order to isolate K⁺ currents, Na⁺ currents were inactivated by 70 ms prepulses to -40 mV while I_Ca,L was blocked by adding 0·3 mM CdCl₂ extracellularly. I_to was defined as the difference between the peak outward current and the sustained component remaining at the end of the depolarizing pulse (I_to = I_peak - I_sus) (Apkon & Nerbonne, 1991). The mean current-voltage relationship for I_to is illustrated in Fig. 3B. I_to densities were reduced at +60 mV from 19·7 ± 1·1 pA pF^-1 (n = 42/10) for sham cells compared with 11·0 ± 1·3 pA pF^-1 (n = 21/6) for post-MI myocytes.

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Figure 3. Ito and Isus in right ventricular myocytes following myocardial infarction A, traces of the transient outward (Ito) and sustained (Isus) current densities in a sham and a post-MI myocyte elicited by 500 ms voltage steps over the range -30 to +70 mV in +10 mV increments from a holding potential of -80 mV (protocol shown in inset, values in mV). Arrows indicate 0 pA pF-1. Ito (B) and Isus (C) were normalized to membrane capacitance and plotted against the test potential for sham-operated (, n = 42/10) and post-MI (, n = 21/6) hearts. Myocytes were depolarized every 5 s. *P < 0·05 between sham and post-MI myocytes at +60 mV.

In contrast to the changes in I_to, the current remaining at the end of a 500 ms depolarizing pulse (I_sus) was not statistically different (P = 0·09) between groups as summarized in Fig. 3C. I_sus density measured at +60 mV was 8·1 ± 0·5 pA pF^-1 (n = 42/10) and 6·7 ± 0·6 pA pF^-1 (n = 21/6) in sham and post-MI myocytes, respectively. The molecular correlate of I_sus remains uncertain, but three K⁺ channel genes (Kv1.2, Kv1.5 and Kv2.1) known to encode for delayed rectifier-type currents have been studied previously. Expression at the transcriptional level of Kv1.2, Kv1.5 and Kv2.1 was not different between sham and post-MI right ventricles (data not shown). RNase protection assays for Kv1.2, Kv1.5 and Kv2.1 were reduced by 16·0 ± 9·2 % (n = 7/7), 20·1 ± 8·9 % (n = 7/7) and 20·4 ± 10 % (n = 7/7) in the post-MI group relative to the sham group (P > 0·05).

Since cardiac hypertrophy and disease have previously been shown to switch gene expression from an adult to a fetal-like program with respect to contractile proteins (Orenstein et al. 1995) we investigated whether the I_to in post-MI myocytes showed features resembling those observed in neonatal myocytes. Previous studies have demonstrated that I_to in neonatal myocytes has a sizeable component that recovers slowly from inactivation (Kilborn & Fedida, 1991; Wickenden et al. 1997). Figure 4A shows typical recordings in a sham and a post-MI myocyte using a double pulse protocol (from -80 to +60 mV) designed to measure the rate at which I_to channels recover from inactivated to closed conformations. Figure 4B graphically depicts the mean recovery data for all sham and post-MI myocytes. For each myocyte, the time course of recovery was fitted using mono- and biexponential functions (see Methods). For sham myocytes (n = 36/10), _fast was 30·1 ± 1·7 ms and accounted for 99·7 ± 0·2 % of the current. For post-MI myocytes (n = 16/8), _fast was 35·7 ± 4·5 ms and accounted for 89·4 ± 2·2 % of the current while _slow was 1686·0 ± 265·3 ms and accounted for 10·6 ± 2·2 % of the current. In spite of the changes in the recovery from inactivation, steady-state activation and inactivation relationships between the groups were not altered significantly (P > 0·2). The mid-points for steady-state activation (i.e. V_½) were 17·1 ± 0·8 mV (n = 42/10) and 16·3 ± 1·5 mV (n = 21/6) while the slope coefficients for activation were 16·7 ± 1·3 and 16·3 ± 1·3 in sham and post-MI myocytes, respectively. The mid-points for I_to inactivation were -31·4 ± 0·7 mV (n = 6/3) and -33·5 ± 1·3 mV (n = 6/3) with the slope coefficients measuring 3·3 ± 0·1 and 3·4 ± 0·2 in sham and post-MI myocytes, respectively.

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Figure 4. Biophysical properties of Ito in right ventricular myocytes following myocardial infarction A, normalized recovery from inactivation traces in a sham and a post-MI myocyte. Arrows indicate 0 pA pF-1. B, plot of recovery kinetics for sham (, n = 36) and post-MI (, n = 14) myocytes using a two pulse protocol (inset) with two identical depolarizing pulses from -80 to +60 mV applied every 10 s at selected intervals from 10 to 10 000 ms. The recovery kinetics in the sham myocyte were best described by a monoexponential function, whereas the recovery kinetics in post-MI myocytes were best fitted by a biexponential function.

The changes in I_to were further investigated by measuring mRNA levels of three K⁺ channel genes, Kv4.2, Kv4.3 and Kv1.4, which are known to encode for I_to-like currents in the rat ventricle (Dixon & McKinnon, 1994; Dixon et al. 1996). Figure 5A shows representative gels of RNase protection assays along with cyclophilin which was used as the internal standard to account for variable loading and possible RNA degradation between experiments (Gidh-Jain et al. 1996). Figure 5A demonstrates a reduction in the mRNA levels of Kv4.2 by 30·3 ± 10 % (n = 7/7) and Kv4.3 by 20·4 ± 5·1 % (n = 7/7), while Kv1.4 mRNA levels were increased by 23·4 ± 3·2 % (n = 7/7) in post-MI hearts compared with sham-operated hearts (P < 0·05). The pattern of mRNA expression for post-MI hearts is similar to that observed in 1- to 2-day-old neonatal hearts (Wickenden et al. 1997).

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Figure 5. Representative comparison of candidate genes encoding the transient outward current in the right ventricle following myocardial infarction A, typical mRNA measurements for Kv4.2 (left panel), Kv4.3 (middle panel) and Kv1.4 (right panel) from sham (left lanes) and post-MI (right lanes) right ventricles. The upper and lower bands represent Kvx- and cyclophilin-protected mRNA fragments, respectively. The bar graphs show mean changes in Kvx mRNA levels in post-MI (, n = 7/7) relative to sham (, n = 7/7). B, Western blot analysis showing immunoreactive proteins for Kv4.2 and Kv1.4 from sham (left lanes) and post-MI (right lanes) right ventricles. The bar graphs show mean changes in Kvx mRNA levels in post-MI (, n = 6/6) relative to sham (, n = 6/6).

To determine whether the changes in mRNA level were accompanied by changes at the protein level, we performed Western blot analysis. Figure 5B shows representative gels of Western blots for Kv4.2 and Kv1.4 from post-MI (n = 6/6) and sham (n = 6/6) controls. Densitometric measurements of Kv4.2 immunoreactive protein for post-MI membrane preparations revealed a 46·0 ± 8·5 % decrease relative to sham preparations (P < 0·05). In contrast, Kv1.4 protein levels were increased by 94·6 ± 42·8 % in the post-MI group relative to the sham group (P = 0·05). The decrease in Kv4.2 correlated with the decreases in I_to density (Fig. 3B). While the increase in Kv1.4 was correlated with the appearance of a slow component of recovery from inactivation in post-MI myocytes.

Action potential duration and [Ca²⁺ ]_i

Action potential prolongation has previously been associated with increased incidences of arrhythmias in rats following MI (Qin et al. 1996). In principle, prolongation of the action potential duration is also expected to elevate [Ca²⁺]_i transients in the myocyte by increasing Ca²⁺ entry via the L-type calcium current (Bouchard et al. 1995). In the absence of 5 mM EGTA and in the presence of [Ca²⁺]_i transients (see Methods), action potentials were also prolonged at 50 % (sham 17·6 ± 2·8 ms (n = 13/5) versus post-MI 34·9 ± 4·0 ms (n = 14/5), P < 0·01) and 90 % repolarization (sham 92·3 ± 15·4 ms (n = 13/5) versus post-MI 633·9 ± 130·1 ms (n = 14/5), P < 0·01) (Fig. 6). The action potential duration in these experiments differed from those illustrated in Fig. 1 because of the contribution of the sodium-calcium exchange current (I_Na-Ca) when [Ca²⁺]_i transients are present. Mean systolic [Ca²⁺]_i was significantly (P < 0·001) elevated in post-MI myocytes (1085·3 ± 112·3 nM, n = 13/7) compared with sham myocytes (401·1 ± 115·0 nM, n = 12/6) while diastolic [Ca²⁺]_i was not significantly (P = 0·11) different between the groups (sham 78·4 ± 22·6 nM versus post-MI 94·6 ± 5·5 nM).

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Figure 6. Action potential and [Ca2+]i characteristics recorded from sham and post-MI right ventricular myocytes A, representative action potentials (upper traces) and [Ca2+]i (lower traces) derived from a sham and a post-MI myocyte under current-clamp conditions. Arrows indicate 0 nM [Ca2+]i and horizontal bars indicate 0 mV. B, mean values for diastolic and systolic [Ca2+]i in sham (, n = 13/8) and post-MI (, n = 13/7) myocytes. *P < 0·05 for systolic [Ca2+]i between sham and post-MI myocytes.

To assess whether changes in action potential profile were responsible for the elevated [Ca²⁺]_i transients, we performed action potential voltage-clamp experiments. Figure 7A shows a typical experiment in which a sham myocyte was stimulated with a typical short sham action potential (VC_Sham) or a representative long action potential from a post-MI myocyte (VC_Post-MI) while Fig. 7B shows the converse experiment in a post-MI myocyte. Peak systolic [Ca²⁺]_i was significantly elevated when sham myocytes were stimulated with a post-MI action potential compared with when stimulated with their own intrinsic action potentials (VC_Sham 283·3 ± 44·1 nM versus VC_Post-MI 813·3 ± 18·6 nM, n = 3/3, P < 0·05) but diastolic [Ca²⁺]_i remained unchanged (VC_Sham 79·0 ± 4·7 nM versus VC_Post-MI 90·0.3 ± 9·0 nM, n = 3/3, P = 0·33). Similar experiments in post-MI myocytes revealed the opposite pattern. Systolic [Ca²⁺]_i was reduced when post-MI myocytes were stimulated with a sham action potential (VC_Post-MI 982·1 ± 184·1 nM versus VC_Sham 333·3 ± 159·2 nM, n = 3, P = 0·05). Again, there was no detectable change in diastolic [Ca²⁺]_i (VC_Post-MI 92·0 ± 7·6 nM versus VC_Sham 88·3 ± 4·4 nM, n = 3/3, P = 0·70). These results demonstrate that [Ca²⁺]_i recorded in sham myocytes with a post-MI action potential command waveform were not significantly different compared with post-MI myocytes stimulated with their intrinsic action potential.

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Figure 7. Effect of action potential prolongation on [Ca2+]i in right ventricular myocytes Action potential voltage-clamp measurements in a sham and a post-MI myocyte. In A, the left panel shows record from a sham myocyte (continuous trace) stimulated with its action potential (dashed trace). The right panel shows the same myocyte stimulated with an action potential derived from a post-MI myocyte (same as that shown in B, left panel). In B, the left panel shows a post-MI myocyte stimulated with its action potential. The right panel shows the same myocyte stimulated with an action potential derived from a sham myocyte. Arrows indicate 0 nM [Ca2+]i and horizontal bars indicate 0 mV.

The results above suggest that elevated [Ca²⁺]_i transients primarily arise from action potential prolongation. To assess this further, we measured [Ca²⁺]_i transients in the two groups under voltage-clamp conditions following a brief 100 ms depolarization step to +10 mV from a holding potential of -80 mV applied at 0·25 Hz. Figure 8A shows representative [Ca²⁺]_i recordings in a sham and a post-MI myocyte. The summarized data are shown in Fig. 8B. The difference in the peak systolic [Ca²⁺]_i between sham and post-MI myocytes disappeared when myocytes were stimulated with identical square voltage waveforms (sham 648·9 ± 143·3 nM (n = 10/6) versus post-MI 736·8 ± 100·8 nM (n = 10/4), P > 0·2). The diastolic [Ca²⁺]_i in sham (91·7 ± 3·0 nM) and post-MI (84·7 ± 3·2 nM) myocytes was also not significantly different (P = 0·14). Furthermore, under our recording conditions, there was no apparent difference in the rate of relaxation of the [Ca²⁺]_i transient between the two groups (data not shown). These results establish that reductions in I_to density are associated with action potential prolongation which is directly responsible for elevated [Ca²⁺]_i transients in post-MI myocytes.

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Figure 8. Effect of short voltage-clamp pulses on the [Ca2+]i transient in sham and post-MI right ventricular myocytes A, representative [Ca2+]i transients in a sham and a post-MI myocyte stimulated with short voltage-clamp pulses (100 ms) from a holding potential of -80 to +10 mV at 0·25 Hz. Arrows indicate 0 nM [Ca2+]i and horizontal bars indicate 0 mV. B, mean values for systolic and diastolic [Ca2+]i in sham (, n = 10/5) and post-MI (, n = 10/4) myocytes. No significant difference was observed between sham and post-MI myocytes.

Depolarizing currents

The changes in APD observed following MI might also originate from an increase in L-type Ca²⁺ currents (I_Ca,L) as observed in other models of heart disease (Keung, 1989; Ryder et al. 1993). Figure 9A shows representative cadmium-subtracted I_Ca,L traces recorded from a sham and a post-MI myocyte in response to 500 ms step depolarizations from -60 mV to +70 mV while holding at -80 mV. On average, Fig. 9B establishes that a small, but non-significant, reduction in I_Ca,L density (P > 0·05 at +10 mV) occurred in post-MI myoctes which goes in the wrong direction to contribute to action potential prolongation.

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Figure 9. ICa,L in right ventricular myocytes following myocardial infarction A, representative current traces showing cadmium-sensitive difference current densities (0·3 mM CdCl2) from a sham and a post-MI myocyte elicited by 500 ms voltage steps to -40, 0, +10 and +30 mV from a holding potential of -80 mV. A prepulse to -40 mV (100 ms) was used to eliminate any contaminating sodium current. Arrows indicate 0 pA pF-1. B, ICa,L density is plotted against the test potential for sham (, n = 15/4) and post-MI (, n = 9/3) myoctes. Myocytes were depolarized every 5 s. No significant difference was observed between sham and post-MI myocytes.

The results above demonstrate that I_Ca,L density is unchanged in post-MI myocytes. However, increases in the net influx of Ca²⁺ as a result of action potential prolongation could occur and thereby contribute to the observed elevations in [Ca²⁺]_i transient amplitude. Therefore, we examined I_Ca,L in action potential voltage-clamp in conditions that eliminated Na⁺, K⁺ and Na⁺-Ca²⁺ exchange currents (see Methods). Figure 10A shows typical cadmium-subtracted Ca²⁺ currents recorded at steady-state in a sham myocyte stimulated with an action potential waveform derived from sham (left panel) or post-MI (right panel) myocytes. The waveforms were applied randomly on the same cell followed by cadmium wash to block I_Ca,L. The post-MI waveform caused a decrease in the peak I_Ca,L, with a marked slowing of its decline (inactivation). The integral of I_Ca,L, which provides a direct measure of calcium entry as a function of time (Bouchard et al. 1995), was increased almost 2-fold in sham myocytes given a post-MI action potential compared with sham action potentials. Table 2 summarizes data for the steady-state Ca²⁺ currents obtained from seven sham myocytes and establishes that action potential prolongation in post-MI myocytes causes a large increase in Ca²⁺ entry per beat.

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Figure 10. Effect of action potential duration on ICa,L The records and graph show ICa,L produced under action potential voltage-clamp conditions. A, recordings from a right ventricular sham myocyte stimulated with representative sham () and post-MI () action potentials (upper traces) and corresponding cadmium-subtracted ICa,L traces (lower traces). Arrows indicate 0 pA and horizontal bars indicate 0 mV. B, current-voltage plot shows ICa,L during the action potential voltage-clamp for a sham () and a post-MI () action potential waveform. Arrows indicate increasing time.

Table 2. Effect of action potential duration on the L-type calcium current

Figure 10B shows a plot of I_Ca,L as a function of voltage during the course of an action potential. The trajectory of the I_Ca,L-voltage relationship when a post-MI action potential is applied follows very closely the relationship shown in Fig. 10B following step depolarizations with the peak occurring at about +10 mV. In contrast, the I_Ca,L-voltage relationship following application of the sham action potential is shifted leftward relative to the post-MI action potential with a peak at about -25 mV. Moreover, a substantial amount of current is observed at voltages below -30 mV which is well below the threshold for L-type Ca²⁺ channel activation. These results strongly suggest that the rate of membrane repolarization in the sham myocyte occurs more rapidly than the rate of channel deactivation similar to that observed in tail current measurements (McDonald et al. 1994). These results suggest that the kinetic nature of Ca²⁺ entry through the L-type Ca²⁺ channels is distinctly changed by action potential prolongation as seen in post-MI myocytes.

SR Ca²⁺ content

While prolonged action potentials clearly elevate [Ca²⁺]_i and increase calcium entry via I_Ca,L, there could also be changes in the Ca²⁺ handling by the sarcoplasmic reticulum in post-MI myocytes. In order to test for this, we measured SR Ca²⁺ content by applying identical step depolarizations (100 ms) under voltage-clamp conditions and integrating Na⁺-Ca²⁺ tail currents following application of 10 mM caffeine. Figure 11A shows the inward currents recorded in response to application of 10 mM caffeine in a sham and a post-MI myocyte with the mean data shown in Fig. 11B. SR Ca²⁺ contents in sham and post-MI myocytes were 97·8 ± 4·9 µM (n = 9/3) and 96·8 ± 19·2 µM (n = 6/3), respectively (P > 0·05), establishing that no differences in SR Ca²⁺ handling exist between the two groups.

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Figure 11. Ca2+ content in the sarcoplasmic reticulum in right ventricular myocytes following myocardial infarction A, original records representing INa-Ca tails following application of 10 mM caffeine in a sham and a post-MI myocyte. All myocytes were held at -70 mV throughout. Dotted traces show non-exchange (leakage) current flowing during the sodium-free portion of caffeine application. Bars indicate the period of caffeine application. B, sarcoplasmic reticulum Ca2+ content was calculated by integrating the currents and the mean data are shown. No significant difference in sarcoplasmic reticulum Ca2+ content was observed between sham () and post-MI () myocytes.

	DISCUSSION

Top Abstract Introduction Methods Results Discussion References

This study was designed to investigate the ionic and molecular basis of action potential prolongation in cardiac hypertrophy and to test if these electrical changes could be linked to alterations in Ca²⁺ handling. Our studies were performed in right ventricular myocardium derived from hearts 8 weeks following left anterior descending coronary artery ligation. As reported previously, both the right and left ventricles of hearts with left-sided infarctions undergo significant compensatory cardiac hypertrophy (Afzal & Dhalla, 1992). Numerous studies have previously documented the pathological, functional, electrical and ionic changes in the left ventricle (Orenstein et al. 1995; Zhang et al. 1995; Kääb et al. 1996; Qin et al. 1996; Rozanski et al. 1998). Few studies have examined changes in the right ventricle following coronary artery ligation which also undergoes significant hypertrophy and remodelling when the infarct size is greater than 35 % of the left ventricular free wall (Table 1). Moreover, functional, biochemical and genetic changes have previously been reported in the right ventricle, which are distinct from changes in the left ventricle of left-infarcted rats hearts (Afzal & Dhalla, 1992; De Tombe et al. 1996; Sethi et al. 1997). In addition, right ventricular diastolic dysfunction is an important predictor of prognosis and mortality in human patients (Yu et al. 1996).

Ionic and molecular mechanisms of action potential duration prolongation

Our data reveal that action potential prolongation occurs in right ventricular myocytes 8 weeks following infarction. The action potentials were significantly prolonged whether in the presence of EGTA (5 mM) in the pipette to minimize the Na⁺-Ca²⁺ exchange activity or under conditions where the Na⁺-Ca²⁺ exchanger is fully operational in the presence of [Ca²⁺]_i transients. These results suggest that K⁺ channel down-regulation is the primary determinant of action potential prolongation.

Underlying these APD changes in post-MI myocytes, I_to density was reduced which correlated with reductions in Kv4.2 mRNA and protein levels and Kv4.3 mRNA levels; two genes known to encode for transient outward currents. Reductions in I_to density and Kv4.2 mRNA and protein was also observed in rat left ventricle 3-4 weeks following infarction (Qin et al. 1996). Interestingly, the level of Kv1.4 mRNA was increased despite reductions in I_to. These results are similar to changes in left ventricular Kv1.4 mRNA expression in spontaneously hypertensive rats (Matsubara et al. 1993), but differ from the nearly 2-fold reduction in Kv1.4 mRNA observed in the left ventricle 3-4 weeks following infarction (Gidh-Jain et al. 1996). These results may indicate temporal or regional differences in the regulation of K⁺ channel expression in diseased hearts. The increase in Kv1.4 mRNA and protein coincided with the emergence of a slow component of I_to recovery kinetics similar to that reported in end-stage human heart failure (Näbauer et al. 1993). The time constant for the slow component is very similar to that recorded previously in Xenopus oocytes expressing Kv1.4 channels (Tseng-Crenk et al. 1990).

The prolonged action potential duration and slowed recovery kinetics of I_to following infarction coincides with that reported in neonatal myocytes (Kilborn & Fedida, 1991; Wickenden et al. 1997). Moreover, the finding that Kv4.2 and Kv4.3 mRNA levels decreased while Kv1.4 increased, is consistent with the re-expression of a fetal electrophysiological phenotype in rat heart, since the reverse changes are known to occur with post-natal development in the rat heart (Kilborn & Fedida, 1991; Wickenden et al. 1997). These results show that switching to a 'fetal gene program' also occurs in the electrophysiological phenotype and K⁺ channel expression as observed for other cardiac genes in diseased hearts (Schwartz et al. 1986). The significance of the re-expression of an early phenotype is unclear. However, as is the case for other aspects of re-expression of the fetal phenotype (Orenstein et al. 1995), these changes might reflect an attempt to maintain contractile performance and hypertrophy (Wickenden et al. 1998) in diseased myocardium.

I_sus was not significantly reduced in post-MI myocytes compared with sham myocytes. Expression at the transcriptional level of Kv2.1, Kv1.5 and Kv1.2 mRNA was slightly decreased (P > 0·05) in post-MI myocytes. These three channel genes encode for currents with delayed rectifier properties (Swanson et al. 1990), but their functional correlation in the rat heart remains uncertain. These changes are similar to those observed in the left ventricle 3-4 weeks following MI (Gidh-Jain et al. 1996).

While reductions in I_to density explain, in large part, the prolongation of the action potential duration, reductions in I_K1 density were also observed. Since, this current contributes to the late phase of repolarization, reductions in I_K1 could further exacerbate action potential prolongation. Indeed, the degree of prolongation at 90 % repolarization exceeded from that observed at 50 % repolarization. In addition, the decrease in I_K1 might also explain the higher resting membrane potentials observed in post-MI myocytes compared with sham myocytes although differences in other background currents cannot be ruled out. For example, a recent report in rat ventricular myocytes has demonstrated an increase in a volume-regulated chloride conductance in response to left ventricular hypertrophy induced by aortic banding (Benitah et al. 1997). Our results are consistent with previous studies showing depolarization of the resting membrane potential (Bouron et al. 1992) and reductions in I_K1 density in diseased hearts (Beuckelmann et al. 1993; Kääb et al. 1996). Despite the decrease in the density of I_K1, expression of IRK1 and IRK2 mRNA was unaltered. These findings raise the possibility that I_K1 expression is downregulated at the post-transcriptional or post-translational level during the development of cardiac hypertrophy.

The role of action potential duration prolongation on [Ca²⁺]_i transients

Previous studies of cardiac disease have demonstrated increases (Brooksby et al. 1993), decreases (Beuckelmann et al. 1992; Cheung et al. 1994) or no change (Cheung et al. 1994) in the [Ca²⁺]_i transient amplitude. In general, the direction of [Ca²⁺]_i transient changes depends on the severity and stage of the disease (Wickenden et al. 1998). Our current-clamp recordings in right ventricular myocytes establish that infarction is associated with prolonged action potential durations and elevations in peak systolic [Ca²⁺]_i without changes in the diastolic [Ca²⁺]_i under our experimental conditions. In action potential voltage-clamp experiments, the mean peak [Ca²⁺]_i transient amplitude was increased 3-fold when sham myocytes were stimulated with an AP waveform derived from a post-MI rat compared with their own shorter action potentials. Conversely, peak [Ca²⁺]_i transients were reduced when post-MI myocytes were stimulated with sham action potentials compared with their intrinsic long action potentials. Importantly, the amplitudes of the [Ca²⁺]_i transients observed in sham myocytes were identical to those observed in the post-MI myocytes stimulated with sham action potentials. On the other hand, [Ca²⁺]_i transient amplitudes in post-MI myocytes were identical to those observed in sham myocytes stimulated with post-MI action potentials. Differences in peak systolic [Ca²⁺]_i were abolished when identical voltage-clamp pulses were delivered to sham and post-MI myocytes, as observed previously in the spontaneously hypertensive rat (Brooksby et al. 1993). These results establish that prolongation of action potential duration is directly responsible for the elevations in [Ca²⁺]_i transient amplitudes observed in the post-MI myocytes. Consistent with this conclusion, the sarcoplasmic reticulum (SR) calcium content was unchanged between the two groups when estimated using I_Na-Ca recordings following application of caffeine. The absence of differences in SR Ca²⁺ content was also previously observed in rats subjected to aortic banding (McCall et al. 1998) and are consistent with previous SR vesicle studies in the rat infarction model (Afzal & Dhalla, 1992). Our results suggest that early in cardiac disease and hypertrophy, action potential duration is prolonged and [Ca²⁺]_i transient amplitude is elevated. In more advanced stages of heart disease and failure, there is consistently a decrease in [Ca²⁺]_i transient magnitude in spite of marked action potential prolongation which is associated with severe reduction in sarcoplasmic reticular Ca²⁺-ATPase and/or Na⁺-Ca²⁺ exchange activity (Beuckelmann et al. 1995). Thus it appears that the changes in systolic [Ca²⁺]_i in the right ventricle that we observed are more representative of the early events occurring during compensated cardiac hypertrophy. The finding that intracellular calcium is elevated has a number of important implications for the pathogenesis and progression of heart failure. The increase in intracellular calcium may initially serve as a compensatory mechanism to increase myocardial contractility of the compromised heart. However, chronic elevations in [Ca²⁺]_i transient amplitude could also contribute to cardiac growth and disease progression by modulating gene expression, since Ca²⁺ is an important cofactor for growth factor signal transduction (Molkentin et al. 1998; Wickenden et al. 1998).

Calcium influx through L-type calcium channels

Unlike I_to and I_K1, I_Ca,L density was not altered in post-MI myocytes compared with sham controls (Fig. 9). These results are consistent with earlier electrophysiological recordings in left ventricle of rat hearts following infarction (Qin et al. 1996) of dog hearts following pacing-induced heart failure (Kääb et al. 1996) and in human heart failure (Beuckelmann et al. 1991). Action potential voltage-clamp recordings did, however, reveal an increase in calcium entry through the L-type Ca²⁺ channels when sham myocytes were stimulated with post-MI action potentials as opposed to sham action potentials (Table 2). This net increase in Ca²⁺ entry in each cardiac cycle is likely to contribute significantly to the increase in [Ca²⁺]_i observed in the post-MI myocytes. Our results establish for the first time that while channel density may be unaffected in cardiac hypertrophy, calcium entry through the channel can increase secondarily via the modulation of hyperpolarizing K⁺ channels and subsequent action potential prolongation.

Interestingly, short action potentials evoked peak I_Ca,L densities which were 2-fold larger and declined much faster than that seen with prolonged action potentials. This appeared to result from a lack of equilibration between channel deactivation and voltage during the rapid repolarization in sham action potentials probably causing a large increase in driving force for Ca²⁺ entry similar to that seen in typical tail experiments (McDonald et al. 1994). These kinetic differences in the nature of Ca²⁺ entry through the L-type Ca²⁺ channels in sham compared with post-MI myocytes may have important implications on the nature of excitation-contraction coupling in cardiac hypertrophy and this is currently being investigated further.

In summary, we report that the right ventricle undergoes hypertrophy, I_to down-regulation and action potential prolongation similar to earlier findings in the left ventricle (Qin et al. 1996). We also found a marked slowing of the recovery kinetics of I_to channels which was correlated with elevation in Kv1.4 mRNA and protein levels. Action potential prolongation following infarction is tightly connected to elevations in peak [Ca²⁺ ]_i via effects on I_Ca,L. Reduction of K⁺ channel expression while arrhythmogenic, also elevates [Ca²⁺]_i, which may be important in regulating contractility, cardiac hypertrophy and disease progression.

	REFERENCES

Top Abstract Introduction Methods Results Discussion References

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Acknowledgements

This study was supported by a grant from the Heart and Stroke Foundation of Ontario. R. K. holds an MRC Doctoral Research Award and P. H. B. is an MRC scholar. Funds for equipment purchases from the Tiffen Trust Fund and the Centre for Cardiovascular Research at the University of Toronto are also acknowledged. The authors wish to thank Dr Fayez Dawood for performing all the required surgery. Special thanks are also extended to Ms Tin Nguyen for performing the RNase protection assays and the Western blot analyses.

Corresponding author

P. H. Backx: Departments of Medicine and Physiology, University of Toronto, 101 College Street, Charlie Conacher Research Wing 3-802, Toronto, Ontario, Canada M5G 2C4.

Email: p.backx{at}utoronto.ca

Author's present address

A. D. Wickenden: ICAgen Inc., 4222 Emperor Boulevard, Ste. 460, Durham, NC 27703, USA.

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