Cardiovascular Research

Cardiovascular Research 1998 40(2):322-331; doi:10.1016/S0008-6363(98)00133-3
© 1998 by European Society of Cardiology

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Copyright © 1998, European Society of Cardiology

Regional differences in the delayed rectifier current (I_Kr and I_Ks) contribute to the differences in action potential duration in basal left ventricular myocytes in guinea-pig

Simon M. Bryant^a,*, Xiaoping Wan^a, S.Jane Shipsey^b and George Hart^a

^aDepartment of Cardiovascular Medicine, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DU, UK
^bDepartment of Cardiology, The London Chest Hospital, Bonner Road, London, UK

^* Corresponding author. Tel.: +44 (1865) 22 01 33; Fax: +44 (1865) 22 19 77.

Received 10 December 1997; accepted 14 April 1998

	Abstract

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
Objective: To compare the properties of single myocytes isolated from different layers of the basal region of the left ventricle and to test the hypothesis that differences in the delayed rectifier current (I_K) contribute to regional differences in action potential duration. Methods: Myocytes were isolated from basal sub-endocardial, mid-myocardial and sub-epicardial layers of the guinea-pig left ventricle. Membrane voltage and current were measured using the switch-clamp technique. Results: Mean action potential duration measured at 90% repolarisation (APD₉₀) was longer in sub-endocardial myocytes than in mid-myocardial and sub-epicardial myocytes [APD₉₀ ms at 0.2 Hz: sub-endocardial 292±12 (n=40), mid-myocardial 243±8 (n=42) and sub-epicardial 227±9 (n=36), P<0.001, analysis of variance (ANOVA)]. The APD–rate relationship (stimulation frequencies 2, 1, 0.2 and 0.017 Hz) was steeper in sub-endocardial than in mid-myocardial or sub-epicardial myocytes (P<0.001, ANOVA). The density of I_K was greater in mid-myocardial (4.05±0.09 pA pF^–1) and sub-epicardial (3.90±0.41 pA pF^–1) than in sub-endocardial myocytes (2.74±0.27 pA pF^–1, P<0.01 ANOVA). The rapidly-activating (I_Kr) and slowly-activating (I_Ks) components of I_K were significantly smaller in sub-endocardial than in mid-myocardial or sub-epicardial myocytes. D,L-Sotalol-induced prolongation of APD₉₀ was similar in the three regions studied. Conclusions: There are significant transmural gradients in the electrophysiological properties of myocytes isolated from the base of the left ventricular free wall in guinea-pig. Sub-endocardial myocytes had a longer APD₉₀ attributable in part to a significantly smaller I_K density. We have been unable to identify M cells in the guinea-pig left ventricular free wall.

KEYWORDS Heart; Electrophysiology; Action potential duration; Ventricular myocytes; Guinea-pig; Potassium current; Antiarrhythmic agents

	1 Introduction

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
Regional differences in action potential characteristics between sub-endocardial and sub-epicardial myocardium have been reported in the dog [1–4], cat [5], rabbit [6], rat [7, 8]and human [9]hearts. Although the mechanisms that underlie these differences are unclear, a larger transient outward current (I_to1) density in sub-epicardial than sub-endocardial myocytes may contribute in part to the regional differences in action potential duration (APD) in the dog [3], cat [5], rat [7, 8]and man [10, 11]. Recently we have demonstrated regional differences in APD between basal sub-endocardial and apical sub-epicardial myocytes in the guinea-pig [12, 13]. However, it is unclear whether these regional differences in APD are due to endocardial/epicardial (transmural) or to apex/base (longitudinal) differences. Two studies have examined transmural gradients in APD in the guinea-pig using multicellular preparations, with conflicting results. Although Watanabe et al. [14]found APD to be longer in basal sub-endocardial than basal sub-epicardial tissue, Sicouri et al. [15]reported no significant difference in APD between these two regions.

Much attention has been paid to the properties of myocytes located in the basal deep sub-epicardium of the canine myocardium, "M cells", [1]and a number of consistent electrophysiological differences have been reported that can be used to differentiate M cells from sub-endocardial and sub-epicardial myocytes. Primarily M cells have a longer APD, larger V_max and a steeper APD–rate relationship than sub-endocardial and sub-epicardial myocytes [1–4]. Indeed Sicouri and Antzelevitch [2]state that "the hallmark of the M cell is the ability of its action potential to prolong disproportionately to that of epicardium and endocardium at slow rates" [2]. Alternative criteria used to distinguish M cells from sub-endocardial and sub-epicardial myocytes are a smaller delayed rectifier current (I_K) density [4], in particular a smaller slowly-activating component (I_Ks) of I_K and an increased sensitivity to class III antiarrhythmics [15, 16]. Although recent studies have suggested that M cells are present in human [9]and guinea-pig [15]hearts they have not been identified in porcine [17]or rat [7]hearts.

The aims of this study were twofold, firstly to test the hypothesis that there are transmural gradients in APD in the guinea-pig left ventricle and whether regional differences in I_K could contribute to such regional differences in APD, and secondly, using the specific criteria utilised to differentiate M cells from sub-endocardial and sub-epicardial myocytes in other species to try to confirm the presence of M cells in the guinea-pig left ventricle.

	2 Methods

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No 85-23, revised 1996).

2.1 Cell isolation
Single myocytes were isolated from discrete regions using a standard enzymatic dispersion technique as described previously [12]. The heart was cannulated on a Langendorff apparatus and perfused for 4 min with a nominally Ca²⁺-free solution followed by a further 5 min with an enzyme-containing solution. The left ventricular free wall was dissected free and tissue samples were dissected from sub-endocardial, sub-epicardial and deep sub-epicardial (mid-myocardial) layers at the base of the ventricle using fine scissors. Tissue samples were then placed in separate flasks containing fresh enzyme solution (with no protease added) for a further 5 and 10 min digestion periods. When the cells were harvested they were washed twice in Tyrode solution (for composition see Section 2.4) containing 5 mg ml^–1 bovine serum albumin (Sigma) and resuspended in Dulbecco's modified Eagle medium supplemented with 2 mg ml^–1 Ultraser G^TM (Gibco, Paisley, Scotland). The myocyte suspension was stored at 20±1°C and cells were used within 12 h of isolation.

Two groups of animals were used in this study. For the majority of experiments myocytes were isolated from young guinea-pigs weighing 486±8 g (n=18). For experiments summarised in Fig. 2B (below) cells were isolated from older guinea-pigs weighing 962±38 g (n=5).

View larger version (17K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 Regional differences in the rate-dependent changes in APD90 in myocytes isolated from young and old guinea-pig hearts. Panel A, mean APD90–rate relationship for 42 sub-endocardial (circles), 43 mid-myocardial (triangles) and 36 sub-epicardial (squares) myocytes isolated from young guinea-pigs. APD was longer and rate-dependency was steeper in sub-endocardial than in mid-myocardial or sub-epicardial myocytes. Panel B mean APD90–rate relationship for 24 sub-endocardial (circles), 25 mid-myocardial (triangles) and 24 sub-epicardial (squares) myocytes isolated from a cohort of older guinea-pigs. APD was longer in sub-endocardial than in mid-myocardial or sub-epicardial myocytes at all frequencies and APD90–rate relationship was similar in all three regions. Data are expressed as mean±S.E.M. *P<0.05, **P<0.01, ***P<0.001, 1-way ANOVA at one stimulation frequency.

 
2.2 Electrophysiological techniques
For electrophysiological recordings myocytes were layered onto the floor of a perfusion chamber situated on the stage of an inverted microscope (Diaphot, Nikon, UK). Cells were superfused with a Tyrode solution (for composition see Section 2.4) at a flow-rate of 1–2 ml min^–1 at 35±1°C. Micropipettes were fabricated from borosilicate capillary tubing (GC200TF-15, Clarke, Pangbourne, UK) and pulled on a vertical puller (Narishige, Japan). Membrane voltage and current were measured using the discontinuous single-electrode voltage-clamp technique (Axoclamp-2A and 2B; Axon, USA) in conjunction with high-resistance microelectrodes (15–20 M when filled with 2 M KCl). Analog signals (current and voltage) were digitised by an analog-to-digital converter (CED 1401; Cambridge Electronic Design, UK or Digidata 1200; Axon, USA) and stored on-line to computer for subsequent off-line analysis.

2.3 Experimental protocols
Action potentials were elicited in current-clamp mode by a 1 ms injection of current at frequencies ranging from 0.2 to 2 Hz. (The current level was adjusted to approximately 50% above threshold). Cell membrane capacitance was measured using a voltage ramp protocol [18]. In guinea-pig ventricular myocytes I_K is comprised of two components, a rapidly-activating component I_Kr and a more slowly-activating component I_Ks [19, 20]. However, I_Kr shows inward rectification at potentials more positive to +20 mV [19, 21]which is due in part to rapid inactivation of the channel [22, 23]. Therefore I_K was measured as peak tail currents elicited on repolarisation back to –40 mV after a 1 s step depolarisation to various test potentials ranging from –30 to +60 mV [24]. The switching frequency was set between 4–6 kHz and the settling time was continuously monitored on an oscilloscope (Wavetek 9012) throughout the duration of the experiment. The sampling frequency and gain were adjusted to ensure that the transient decayed completely before the next sample was taken. A holding potential of –40 mV was used to inactivate I_Na. Nicardipine (0.1 µM) was used to block I_Ca and BAPTA (20 mM) was added to the pipette solution to eliminate interference from Ca²⁺-activated currents [24]. Dofetilide (a methanesulfonanilide class III antiarrhythmic drug) was used to specifically block I_Kr at a concentration of 1 µM [21]thus dofetilide-insensitive current was taken to be I_Ks and dofetilide-sensitive current (i.e. I_K–I_Ks) was taken to be I_Kr.

2.4 Drugs and solutions
The composition of the isolation Tyrode solution was as follows (mmol l^–1): NaCl 130; KCl 5.4; MgCl₂ 3.5; NaH₂PO₄ 0.4; glucose 10; 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid (HEPES) 5; CaCl₂ 0.75; pH 7.2 at 35°C with NaOH. The nominally Ca²⁺-free solution contained no added calcium, and EGTA (0.1 mmol l^–1). The enzyme solution contained collagenase (1 mg ml^–1, Worthington, NJ, USA), protease (0.04 mg ml^–1, Sigma) and Ca²⁺ (0.05 mmol l^–1). The composition of the superfusion solution was (mmol l^–1): NaCl 134; KCl 5.4; MgCl₂ 1.2; CaCl₂ 1.8; glucose 11.1; HEPES 5; pH 7.4 with NaOH. D,L-Sotalol (Sotacor, Bristol–Myers) stock solution (32 mmol l^–1) was diluted to 32 µM; dofetilide (a gift from Pfizer Central Research, Pfizer, Sandwich, Kent, UK) stock solution (10 mmol l^–1 dissolved in dimethylsulfoxide) was diluted to 1 µM and nicardipine (Sigma) stock solution (0.1 mmol l^–1 dissolved in pure water) was diluted to 0.1 µM. BAPTA (Sigma) was dissolved directly into 2 M KCl to 20 mmol l^–1.

2.5 Statistics
Data are expressed as mean±standard error of the mean (S.E.M.) and n indicates the number of cells used in each group. Differences between the three regions were assessed using one-way analysis of variance (1-way ANOVA). Current–voltage and mean APD measured at 90% repolarisation (APD₉₀)–rate relationship curves were compared using repeated measures ANOVA. The effects of D,L-sotalol on APD₉₀ were assessed using two-way ANOVA (2-way ANOVA) using region and D,L-sotalol as factors. Individual post hoc comparisons were made using Student–Newman–Keuls test (SNK). Statistical significance was taken at 0.05 level.

	3 Results

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
3.1 Action potential characteristics
Fig. 1 shows representative action potential records taken from individual sub-endocardial (circle), mid-myocardial (triangle) and sub-epicardial myocytes (square) recorded at a stimulation frequency of 0.2 Hz. Although the resting potential is similar in the three cell types the action potential recorded from the sub-epicardial myocyte has a lower amplitude and a shorter duration than either the sub-endocardial or the mid-myocardial myocyte. Mean data for action potential characteristics are shown in Table 1. There are significant transmural differences in the action potential overshoot and amplitude, and in APD at both 50% (APD₅₀) and 90% (APD₉₀) repolarisation levels. Mean values for action potential overshoot and amplitude are smaller in sub-epicardial than in sub-endocardial (10% and 4% respectively) or mid-myocardial myocytes (13% and 5% respectively). Mean APD₉₀ of sub-endocardial myocytes is 20% longer than mid-myocardial and 28% longer than sub-epicardial myocytes. Although there are significant differences in the mean values for action potential overshoot and amplitude between mid-myocardial and sub-epicardial myocytes the differences in APD₅₀ and APD₉₀ between these two regions did not reach statistical significance.

View larger version (12K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 Regional differences in action potential characteristics between basal sub-endocardial (circle), mid-myocardial (triangle) and sub-epicardial (square) myocytes. Representative action potentials were recorded from individual sub-endocardial, mid-myocardial or sub-epicardial myocytes isolated from normal hearts at a stimulation frequency of 0.2 Hz.

 

View this table: [in this window] [in a new window]   Table 1 Regional differences in action potential characteristics in basal ventricular myocytes

 
3.2 Rate-dependent changes in APD
A distinguishing feature of M cells is a marked prolongation of APD associated with a decrease in stimulation frequency. Mean data for APD₉₀ recorded at different stimulation frequencies (0.017, 0.2, 1, and 2 Hz) in sub-endocardial (open circles), mid-myocardial (open triangles) and sub-epicardial myocytes (open squares) are shown in Fig. 2A. APD is significantly longer in sub-endocardial myocytes than mid-myocardial and sub-epicardial myocytes at all three frequencies. Moreover, the rate-dependent change in APD₉₀ is different in the three regions (P<0.001, repeated measures ANOVA). Prolongation of APD elicited by changing the stimulation frequency from 2 to 0.2 Hz is greater in sub-endocardial myocytes (17.3±3.8%) than in mid-myocardial (7.5±1.9%) and sub-epicardial myocytes (8.4±2.2%) (P<0.02, 1-way ANOVA).

It has been suggested that rate-dependent changes in the action potential characteristics increase with age [25]. Fig. 2B shows the mean APD–rate relationships for myocytes isolated from older guinea-pigs. Although APD₉₀ remained longest in the sub-endocardial myocytes (open circles), the rate-dependent changes in APD₉₀ observed were similar in the three groups (P=0.1, repeated measures ANOVA).

3.3 Regional differences in I_K
Regional differences in APD within the canine left ventricle may be explained, at least in part, by regional differences in I_K density [4]. To test the hypothesis that regional differences in I_K density may contribute to the regional differences in APD in the guinea-pig we measured the voltage-dependence of I_K tail currents in the three regions studied. Fig. 3 shows representative families of tail current of records (elicited on repolarisation to –40 mV after a 1 s step depolarisation to various test potentials, see panel B) taken from single sub-endocardial, mid-myocardial and sub-epicardial myocytes. The magnitude of the tail currents elicited in the sub-endocardial myocyte are smaller than those recorded in the mid-myocardial and the sub-epicardial myocyte.

View larger version (26K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 Regional differences in IK tail currents. Panel A, representative IK tail current records taken from individual basal sub-endocardial, mid-myocardial and sub-epicardial myocytes elicited upon repolarisation to –40 mV after a 1 s step depolarisation to –20 mV, 0 mV, +20 mV, +40 mV, and +60 mV. For the sake of clarity in panel A only the last 10 ms of the step depolarisation and the first 500 ms after repolarisation to –40 mV are shown. Panel B, shows an example of the protocol used to elicit time-dependent and tail currents. Panel C, Current–voltage relationship curves for IK tail currents recorded from 12 sub-endocardial (circles), 9 mid-myocardial (triangles) and 12 sub-epicardial (squares) myocytes. Data are expressed as mean±S.E.M. For the sake of clarity error bars are only shown when they are larger than the symbol size. , P<0.05 (SNK) sub-endocardial myocytes are different from both mid-myocardial and sub-epicardial myocytes.

 
Mean current density–voltage relationship curves for I_K recorded under control conditions, for sub-endocardial (open circles), mid-myocardial (open triangles) and sub-epicardial (open squares) myocytes are plotted in Fig. 3C. The current density–voltage relationship for I_K is significantly different in the three regions studied (P<0.001, repeated measures ANOVA, voltage range –30 to +60 mV). I_Ks density is significantly lower in sub-endocardial than both mid-myocardial and sub-epicardial myocytes at voltages positive to –20 mV (see Fig. 3C). The mean values for tail current density elicited on repolarisation to –40 mV after a 1 s step depolarisation to 0 mV and +60 mV are given in Table 2. I_K tail current density is significantly smaller in sub-endocardial than in mid-myocardial or sub-epicardial myocytes.

View this table: [in this window] [in a new window]   Table 2 Regional differences in the delayed rectifier current in basal ventricular myocytes

 
To separate I_K into its individual components [19]we used dofetilide (1 µM) to selectively block I_Kr [21]. Mean current density–voltage relationship curves for the dofetilide-insensitive tail current (I_Ks) and the dofetilide-sensitive tail current (I_Kr) for sub-endocardial (open circles), mid-myocardial (open triangles) and sub-epicardial (open squares) myocytes are plotted in Fig. 4(A and B).

View larger version (21K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4 Regional differences in the mean current density–voltage relationship curves for IKs and IKr. Current–voltage relationship curves for IK tail currents recorded from 12 sub-endocardial (circles), 9 mid-myocardial (triangles) and 12 sub-epicardial (squares) myocytes in the presence of dofetilide (1 µM), IKs (panel A) and the dofetilide-sensitive current, IKr (i.e. IK–IKs panel B). Note that panel B has a different scale. Panel C, activation curves for IKr (open symbols) and IKs (closed symbols) recorded from sub-endocardial (circles, dotted lines), mid-myocardial (triangles, solid lines) and sub-epicardial (squares, dashed lines) myocytes. The plotted curves were calculated by fitting the mean data points to a sigmoid function. The potential at which half activation (V0.5) occurs and the slope factor (k) of the relationship for IKr and IKs are similar in the three regions. IKr: V0.5; endo –18 mV, mid –21 mV and epi –20 mV and k; endo 12 mV, mid 8 mV and epi 10 mV IKs: V0.5; endo +30 mV, mid +24 mV and epi +28 mV and k; endo 13 mV, mid 13 mV and epi 15 mV. Activation of IKs occurs at more positive potentials than IKr in all the three regions studied. Data are expressed as mean±S.E.M. For the sake of clarity error bars are only shown when they are larger than the symbol size. , P<0.05 (SNK) sub-endocardial myocytes are different from both mid-myocardial and sub-epicardial myocytes.

 
Mean current density–voltage relationship for I_Ks is different in the three regions studied (P<0.001, repeated measures ANOVA, voltage range –30 to +60 mV). I_Ks density is significantly lower in sub-endocardial than in mid-myocardial or sub-epicardial myocytes at voltages positive to +20 mV (see Fig. 4A).

Mean I_Kr density–voltage relationship curves are plotted in Fig. 4B and mean values for I_Kr density are given in Table 2. The magnitude of I_Kr tail current elicited on repolarisation to –40 mV after a step depolarisation to 0 mV is different in the three regions studied (P<0.05, 1-way ANOVA). I_Kr density is smaller in sub-endocardial than in mid-myocardial or sub-epicardial myocytes. However, statistically the current density–voltage relationship curves for the three cell types was not different (P=0.8, repeated measures ANOVA, voltage range –30 to +60 mV).

3.3.1 Activation of I_Kr and I_Ks
Voltage-dependent activation of I_K tail currents under control conditions was not monotonic, and it is possible that this may be attributable to the overlapping of the activation curves for the two components of I_K [24]. Therefore we have constructed voltage-dependent activation curves for I_Kr and I_Ks in the three regions studied (Fig. 4C). Though I_Kr and I_Ks activate at widely different voltages, the activation curves for I_Kr or I_Ks were similar in the three regions studied.

3.4 The effects of D,L-sotalol on APD
Fig. 5A shows representative action potential records taken from individual sub-endocardial, mid-myocardial and sub-epicardial myocytes and recorded in the absence and presence of D,L-sotalol (32 µM). Mean values for APD₉₀ are shown in Fig. 5B. APD₉₀ was prolonged by the application of D,L-sotalol (P<0.005, 2-way ANOVA) to the same extent in the three regions (APD₉₀ was prolonged by 17±3% in sub-endocardial myocytes, by 17±4% in mid-myocardial myocytes and by 23±6% in sub-epicardial myocytes, P<0.52, 2-way ANOVA)

View larger version (20K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 5 The effects of D,L-sotalol on APD90. Panel A, representative action potential records taken from individual sub-endocardial, mid-myocardial and sub-epicardial myocytes under control conditions and after 3 min perfusion with D,L-sotalol (32 µM, marked with the solid circle). Stimulation frequency was 0.2 Hz. Panel B, APD90 recorded from 11 sub-endocardial, 14 mid-myocardial and 12 sub-epicardial myocytes in the absence (open bars) and presence (closed bars) of D,L-sotalol. Data are expressed as mean±S.E.M. APD90 is significantly prolonged by the application of D,L-sotalol in all three regions (P<0.001, 2-way ANOVA). APD90 is significantly different in the three regions in the absence and presence of D,L-sotalol (P<0.01, 2-way ANOVA).

 
APD was different in the three regions in the absence and presence of D,L-sotalol (P<0.01, 2-way ANOVA).

	4 Discussion

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
These data demonstrate for the first time transmural differences in the electrophysiological properties of single myocytes isolated from the base of the left ventricle in normal guinea-pig hearts. Sub-endocardial myocytes have a longer APD and a steeper APD–rate relationship than mid-myocardial and sub-epicardial myocytes. The regional differences in APD may be attributable in part to a smaller I_K density in sub-endocardial than in mid-myocardial or sub-epicardial myocytes.

4.1 Regional differences in action potential characteristics
Previous studies have shown that APD is longer in sub-endocardial than in sub-epicardial myocytes in the dog [3, 4], rabbit [6], and rat [7, 8]. However in the guinea-pig, APD of basal sub-endocardial tissue has been reported to be both similar to [15]and longer than [14]that recorded in basal sub-epicardial tissue. Recently mean APD₉₀ recorded in isolated basal sub-endocardial myocytes has been shown to be longer than in apical sub-epicardial myocytes in the guinea-pig [12, 13], though it is not known whether these differences were due to base/apex or to endocardial/epicardial differences.

In this study we show significant transmural gradients in APD in basal ventricular myocytes in the guinea-pig and in contrast to Sicouri et al. [15]who recorded APD in muscle strips obtained from the guinea-pig left ventricle, we found that sub-endocardial myocytes had the longest APD, sub-epicardial myocytes had the shortest and mid-myocardial myocytes had intermediate values of APD (see Fig. 1 and Table 1). These data are in accord with the findings of Watanabe et al. [14]and support the notion that there are transmural gradients in APD in the guinea-pig left ventricle.

The significant regional differences in the overshoot and amplitude of the action potential (and APD₁₀, data not shown) between the three cell types suggest that there are transmural differences in membrane currents that underlie the early part of the action potential such as I_Na (and/or an early outward current) at the base of the guinea-pig left ventricle.

4.2 Regional difference in the rate-dependent changes in APD
Prolongation of APD associated with a decrease in stimulation frequency has been shown to be more pronounced in mid-myocardial myocytes than in sub-endocardial or sub-epicardial myocytes in the dog [1–4]and human [9]. We found a significant regional difference in the rate-dependent changes in APD₉₀ but in contrast to Sicouri et al. [15](who showed that APD–rate relationship was only moderately steeper in mid-myocardial than in sub-endocardial or sub-epicardial myocytes) these data show that APD–rate relationship is steeper in sub-endocardial than in mid-myocardial or sub-epicardial myocytes. Moreover, excessive rate-dependent prolongation of APD (i.e. >100%) reported in mid-myocardial myocytes in other species was not observed in any experiment.

It has been reported that in the canine ventricle M cells do not become electrophysiologically distinct until 3 months of age [26]. It has been suggested that developmental changes in I_to contribute in part to the age related changes in the APD–rate relationship in the canine ventricle. Although I_to is absent in the guinea-pig ventricle [18, 27]M cells have been reported in 8 to 16 week old guinea-pigs weighing 350–800 g [15]. The mean weight of guinea-pigs used in this study was 486±8 g (approximate age 10 –14 weeks). However, to exclude possible effects of maturity we also isolated cells from older animals (>6 months old, 962±38 g). If M cells were developing during this time then one might expect the rate-dependent changes in APD to become different from sub-endocardial and sub-epicardial myocytes. However this appears not to be the case, data from these animals suggest that the APD–rate relationship was similar in the three regions (see Fig. 2B).

4.3 Regional differences in I_K density
Few studies have examined the regional distribution of I_K although it is a major determinant of the time course of repolarisation in the guinea-pig [20]. In the canine ventricle the density of I_K in sub-epicardial myocytes is greater than in mid-myocardial [4, 28]and similar to sub-endocardial myocytes [4]. However, the density of I_K has been shown to be greater in apical sub-epicardial than basal sub-endocardial myocytes in the cat [29]and guinea-pig [30]. These data show a significant transmural gradient in I_K density in basal ventricular myocytes. The regional differences in I_K density correlate closely with the regional differences in APD₉₀, i.e., sub-epicardial myocytes have the shortest APD₉₀ and largest I_K density and sub-endocardial myocytes have the longest APD₉₀ and smallest I_K density. These data suggest that regional differences in I_K contribute to the regional differences in APD.

It is unknown whether the differences in I_K are due to differences in regulation or expression of the channel proteins. Brahmajothi et al. [31]have shown that both mRNA transcript and protein expression of ERG (a channel protein known to play an important role in the generation of a component I_K) were more abundant in the epicardial cell layers than endocardial cell layers in the ferret [31]and thus had a similar regional distribution to I_K. It is interesting to note that in the rat left ventricle both I_to1 density and the expression of Kv4.2 mRNA (a gene which contributes to the I_to1 channel [32]) are greater in epicardium than in endocardium [7, 32, 33].

4.3.1 Regional differences in I_Ks density
In the canine ventricle the density of I_Ks has been shown to be similar in sub-endocardial and sub-epicardial myocytes and to be significantly smaller in mid-myocardial myocytes [4]. It is suggested that the reduced I_Ks density in mid-myocardial myocytes in part explains to properties of M cells in dogs (i.e. the regional differences in APD, APD–rate-dependence and sensitivity to class III antiarrhythmics) [4]. Our data show that in the guinea-pig the density of I_Ks is significantly greater in mid-myocardial and sub-epicardial myocytes than in sub-endocardial myocytes and thus may contribute to the regional differences in APD in basal ventricular myocytes.

4.3.2 Regional differences in I_Kr density
Tail currents evoked upon repolarisation back to –40 mV after a step depolarisation to 0 mV will consist primarily of I_Kr (as I_Kr is selectively activated at 0 mV [24, 34]due in part to differences in the voltage-dependence of activation of I_Kr and I_Ks, see Fig. 5 [24]). Under control conditions (normal Tyrode) we found regional differences in I_K tail current density (elicited on repolarisation back to –40 mV after a step depolarisation to 0 mV). Moreover, we found significant regional differences in the dofetilide-sensitive but not the dofetilide-insensitive tail current (see Table 2). Thus in contrast to the canine ventricle (in which I_Kr has been reported to be similar in sub-endocardial, mid-myocardial and sub-epicardial myocytes [4]) our data suggest that there are regional differences in I_Kr in the guinea-pig and thus the smaller I_K density observed in sub-endocardial myocytes is due in part to lower I_Ks and I_Kr densities.

Although I_Kr activates at different voltages than I_Ks (see Fig. 4C) the voltage at which half-maximal activation (V_0.5) of I_Kr and I_Ks occurs was similar in the three regions. The average V_0.5 values for I_Kr and I_Ks are –19.6 mV and +27 mV respectively which concur with previously reported values [24].

4.4 Regional differences in the effect of D,L-sotalol
M cells are considered to have an increased sensitivity to class III antiarrhythmic compounds compared to sub-endocardial and sub-epicardial myocytes and recently Sicouri et al. [15]showed that the application of D,L-sotalol (100 µM for 10 min) caused a greater prolongation of APD in mid-myocardial myocytes than in sub-endocardial and sub-epicardial myocytes. Moreover, early afterdepolarisations were observed in mid-myocardial but not in sub-endocardial or sub-epicardial myocytes. In contrast and despite possible differences in I_Kr density between the three regions studied, we found that the effect of D,L-sotalol (32 µM) was similar in the three groups. The reason for this apparent discrepancy is unclear. The effects of high concentrations of D,L-sotalol (above 100 µM) may be attributable at least in part to inhibition of I_Na and I_K1 as well as I_Kr [19, 35]. Although it is possible that in this study complete block of I_Kr was not achieved with 32 µM D,L-sotalol [half-maximal inhibition of I_K tail current in rabbit Purkinje fibres was achieved with 10 µM D,L-sotalol [36], and the IC₅₀ value for the inhibition of [³H]dofetilide binding to the high-affinity site (I_Kr) in guinea-pig ventricular myocytes by sotalol is 14 µM [37]], we found that APD₉₀ was prolonged by 19±3% (n=37). Our data are similar to the findings of Sanguinetti and Jurkiewicz [19]who showed that E-4031 (a class III antiarrhythmic agent) caused a 24% prolongation APD in guinea-pig ventricular myocytes. Moreover, Zeng et al. [20]have shown that complete block of I_Kr prolongs APD by 24%, and that this does not induce early afterdepolarisations [20]. In this study early afterdepolarisations were not observed in mid-myocardial myocytes and in one out of 11 sub-endocardial myocytes (data not shown).

In the presence of D,L-sotalol there is still a significant regional difference in APD₉₀, sub-endocardial APD₉₀ being longer than sub-epicardial APD₉₀ (P<0.05, SNK 1-way ANOVA) suggesting that there must be regional differences in membrane currents other than I_Kr. This could reflect the regional difference in I_Ks.

4.5 The presence of M cells in the guinea-pig left ventricle
Although several studies have demonstrated the presence of M cells primarily in the canine [1–4]but also in human [9]and recently guinea-pig [15]myocardium, other studies have found no evidence of M cells in pig [17]or rat [7]hearts. In contrast to the data of Sicouri et al. [15]we found no evidence of M cells in this study and at present the reason for this discrepancy is unclear. It is possible that methodological differences between the multicellular tissue preparations as used by Sicouri et al. [15]and isolated myocytes used in this study may explain our inability to find M cells in the guinea-pig. Factors that influence APD recorded in multicellular preparations during changes in stimulus frequency that are not present in isolated myocytes preparations include cell-to-cell coupling (both mechanical and electrical), changes to extracellular ion concentration (accumulation and/or depletion) and changes to the metabolic status of the cell.

In conclusion our data demonstrate the existence of significant transmural differences in the electrophysiological properties of myocytes isolated from the base of the left ventricular free wall in the guinea-pig. Sub-endocardial myocytes had longer APD, a steeper APD–rate relationship and a smaller I_K density than mid-myocardial and sub-epicardial myocytes. Moreover, the properties of mid-myocardial myocytes in this study did not fulfil any of the criteria used to differentiate M cells from sub-endocardial and sub-epicardial myocytes and thus we have been unable to confirm the presence of M cells in the guinea-pig left ventricular free wall.

Time for primary review 33 days.

	Acknowledgements

 
This work was supported by the British Heart Foundation. Jane Shipsey was a British Heart Foundation Junior Research Fellow. Xiaoping Wan was in receipt of an Overseas Research Studentship.

	References

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 

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