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Cardiovascular Research 2002 55(1):64-75; doi:10.1016/S0008-6363(02)00403-0
© 2002 by European Society of Cardiology
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Copyright © 2002, European Society of Cardiology

Changes in cardiac myocyte morphology alter the properties of voltage-gated ion channels

Kenneth B Walsh* and Graham E Parks

Departments of Pharmacology and Physiology, University of South Carolina, School of Medicine, Columbia, SC 29208, USA

* Corresponding author. Tel.: +1-803-733-3261; fax: +1-803-733-3197 walsh{at}med.sc.edu

Received 20 August 2001; accepted 20 February 2002


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: The goal of this study was to determine if the properties of the transient outward potassium (Ito), TTX-resistant sodium (INa) and L-type calcium (ICa) currents are altered during changes in cardiac cell shape. Methods: Ventricular myocytes were isolated from 3- to 4-day-old neonatal rats and cultured on either non-aligned or aligned collagen thin gels. In contrast to the flat, stellar-shaped myocytes obtained when the cells are plated on non-aligned collagen gels, myocytes plated on aligned gels display an elongated, rod-like shape. Ion channel expression was measured using the whole-cell arrangement of the patch clamp technique and Western blot analysis. Results: Peak values for Ito, INa and ICa were 9±1, 71±13 and 7±1 pA/pF, respectively, in the flat cells, and increased to 21±2, 190±26 and 13±1 pA/pF, respectively, in the aligned cells. Application of forskolin (2 µM) and 3-isobutyl-1-methylxanthine (100 µM) resulted in a 101±18% increase in ICa in the flat cells, but increased the current by only 43±9% in the aligned cells. Internal dialysis of the myocytes with cAMP strongly increased the peak ICa in the flat cells, but caused no significant change in the aligned cells. While both basal and forskolin-stimulated levels of cAMP were the same in the two cell morphologies, the expression of the calcium channel {alpha}1C subunit was increased in the aligned cells. Conclusions: The expression and regulatory properties of voltage-gated calcium channels are modified during changes in neonatal rat myocyte shape.

KEYWORDS Cell culture/isolation; Ion channels; Myocytes; Signal transduction


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Three prominent voltage-gated ion currents are expressed in cardiac ventricular muscle; the tetrodotoxin (TTX)-resistant sodium current (INa), the L-type calcium current (ICa) and the transient outward potassium current (Ito). These currents contribute in a precisely timed and regulated manner to the development, maintenance and termination of the action potential [1]. Recent studies have suggested that voltage-gated ion channels in a number of tissues, including the heart, are targeted to cell membrane locations through interaction with membrane associated proteins such as the syntrophin and PSD-95 families of protein [2–4]. Changes in myocyte structure that would disrupt or enhance this targeting might therefore lead to significant changes in cardiac excitability.

It is clear that cellular function is intimately linked to the phenotype that a given cell type expresses. This interdependency between cell shape and function is epitomized in the structural organization of the terminally differentiated cardiac myocyte. The sarcomeric arrangement of the contractile proteins is carefully aligned through cytoskeletal support proteins and cell adhesion molecules that ensure efficient cardiac contraction [5,6]. Disruption of these support proteins leads to myofibrillar abnormalities, changes in cell shape and perturbations in protein metabolism [7,8].

While short term changes in cardiac myocyte shape, induced by mechanical and osmotic forces, are known to regulate the opening of cardiac ion channels [9–11], the relationship that exists between the overall cardiac cell phenotype and the expression of voltage-gated channels is unknown.

In the present study Ito, INa and ICa were recorded from neonatal rat ventricular myocytes cultured on either non-aligned (random) or aligned collagen-coated silastic membranes. Unlike the flat, stellar-shaped myocytes that become distributed on the non-aligned collagen membranes, myocytes plated on the aligned collagen organize along a common axis and display an elongated, rod-like shape. It is reported that the current densities of Ito, INa and ICa were all increased in the aligned cells. Furthermore, when compared with the non-aligned cells, the stimulation of ICa by protein kinase A was diminished in the aligned myocytes. Thus, changes in neonatal cardiac cell shape alter both ion channel expression and the regulation of voltage-gated calcium channels.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
The investigation conforms to 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) and was in accordance with the institutional guidelines for animal care at the University of South Carolina.

2.1 Isolation and culture of cardiac ventricular myocytes
Neonatal rat ventricular myocytes were isolated and cultured as described previously [12,13]. In brief, heart ventricles were removed from neonatal pups (days 3–4 in age), minced into 1 mm3 pieces and subjected to collagenase (type B, Boehringer Mannheim Biochemicals) dissociation [12]. Following isolation, the cells were plated on either non-aligned (random) or aligned, collagen thin gel-coated silastic membranes (Specialty Manufacturing) [13]. For the preparation of the aligned gels, collagen (type I, Celtrix) was applied to the peripheral edges of the culture dish containing the membranes. While the dish was tilted, a small sterile scraper was used to draw the collagen solution across the silastic membrane. Excess collagen was then aspirated and the culture dish transfered to a 37 °C incubator. When plated on this oriented or aligned collagen substrate, the cells exhibited an in vivo-like phenotype with a rod-shaped appearance (see Fig. 1) [13]. Cells plated on the non-aligned collagen displayed the typical stellar-shaped, or flat morphology described previously (see Fig. 1) [13]. Myocytes were cultured in DMEM (Gibco), supplemented with 10% horse serum (Flow Laboratories) and maintained in a humidified atmosphere of 5% CO2 at 37 °C. Both the aligned and flat cultures displayed spontaneous beating. After allowing 2–3 days for cell attachment, silastic membranes were transferred to a recording chamber for patch clamp measurements. In some experiments, cardiac myocytes were isolated from 4-week-old rat hearts using retrograde aortic dialysis [14]. These cells were stored in 132 mM NaCl external solution (see below) and used within 1–10 h of isolation.


Figure 1
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  		Fig. 1 Neonatal rat ventricular myocytes cultured on aligned and flat collagen thin gels. Myocytes were prepared as described in the Methods section and cultured either on flat (top panel) or aligned (bottom panel) collagen-coated plates.

 
2.2 Recording procedure and measurement of voltage-gated ion currents
The patch clamp method [15] was used to record the whole-cell sodium (INa), L-type calcium (ICa) and transient outward potassium (Ito) currents using L/M EPC-7 (Adams and List Associates) and Axopatch 200 (Axon Instruments) amplifiers. Our procedure for measurement and analysis of these voltage-gated ion currents has been described previously [14,16]. Pipettes were made from Gold Seal Accu-fill 90 Micropets (Clay Adams Inc.) and had resistances of 1–2 M{Omega} when filled with internal solution. All experiments were conducted on isolated, non-coupled myocytes at room temperature (22–24 °C). For the measurement of ICa and Ito, cells were placed in an external solution consisting of (in mM); 132 NaCl, 5 KCl, 2 MgCl2, 1 CaCl2, 5 dextrose, 5 HEPES, pH 7.4 (with NaOH) (280 mOsm.). Ito recordings were made with an internal solution of (in mM); 50 KCl, 60 K-glutamate, 2 MgCl2, 1 CaCl2, 11 EGTA, 5 ATP (K+ salt), 10 HEPES, pH 7.3 (with KOH) ([K+]=140 mM) (280 mOsm). ICa was eliminated by addition of CdCl2 (100 mM) to the external solution. INa was reduced with tetrodotoxin (TTX) (10 or 20 µM). The internal solution for INa and ICa measurements consisted of (in mM); 70 CsCl, 40 Cs–Aspartate, 2 MgCl2, 1 CaCl2, 11 EGTA, 3 ATP (K+ salt), 10 HEPES, pH 7.3 (with CsOH) (280 mOsm.). INa recordings were performed with a reduced sodium external solution that contained (in mM); 100 TEACl, 37 NaCl, 2 MgCl2, 0.5 CaCl2, 0.5 CsCl, 5 dextrose, 5 HEPES, 0.1 CdCl2, pH 7.4 (with CsOH).

In some ICa experiments cAMP (100 µM) and the PKA 6-22 amide inhibitory protein (PKI) (20 µM) were added to the cesium internal solution and dialyzed into the myocytes. After disruption of the cell membrane, chemicals in the internal solution move into the cell by diffusion. Using a simple compartmental model, Kameyama et al. [17] have predicted that substances like cAMP (molecular mass=329, diffusion coefficient=5x10–6 cm2/s) reach 90% of the pipette concentration within 5 min. Larger molecular weight substances, such as PKI (molecular mass=1868), would be expected to reach equilibrium at a slower rate. In order to allow for diffusion of cAMP and PKI into the cells, currents were measured 5–15 min after establishment of the whole-cell configuration.

Membrane currents were recorded with 12-bit analog/digital converters (Axon Instruments). Data were sampled at either 10 (INa and ICa) or 2.5 kHz (Ito) and filtered at 2 (INa and ICa) or 0.5 (Ito) kHz with a low pass Bessel filter (Frequency Devices). Pulse intervals of 2, 5 and 5 s for INa, ICa and Ito, respectively, were designed to allow for interpulse recovery from inactivation. Series resistance was compensated to give the fastest possible capacity transient without producing oscillations. With this procedure >70% of the series resistance could be compensated. Averaged current values presented are means±S.E. Where appropriate, statistical significance was estimated using Student's t-test for unpaired observations.

2.3 Preparation of cardiac cell lysates and Western blot analysis
To prepare cell lysates for Western blot analysis, cardiac myocyte cultures were incubated with a modified RIPA buffer (20 mM Tris–HCl, pH 7.4 and 0.5% Deoxycholate, 0.1% SDS, 0.1% Triton X-100). Lysates were then transferred to microfuge tubes and sonicated to reduce viscosity. Cellular and nuclear debris were removed by centrifugation at 1200xg for 15 min. In order to measure the calcium channel {alpha}1C subunit, a crude membrane fraction was prepared using centrifugation at 100 000xg for 30 min.

The protein content of the cell preparations was determined using a protein assay kit (Pierce). For Western blot analysis, proteins were separated by electrophoresis on either 6% (calcium channel {alpha}1C subunit) or 10% (Kv {alpha} subunits) SDS polyacrylamide gels using a mini PROTEAN cell (Bio-Rad) at 80 V for 2.5 h. The running buffer contained 20 mM Tris, 193 mM glycine, pH 8.3 and 0.1% SDS. Proteins were transferred to polyvinylidene difluoride membranes (Bio-Rad) using a mini Trans-Blot apparatus (Bio-Rad). The transfer buffer contained 25 mM Tris, 192 mM glycine, pH 8.5 and 10% ({alpha}1C subunit) or 20% (Kv {alpha} subunits) methanol. For immunodetection, membranes were first blocked in PBS containing 0.1% Tween-20, 5% Carnation nonfat dry milk and 0.025% Na Azide for 60 min at room temperature. Antibodies to the voltage-gated K+ channel {alpha} subunits (rat anti-Kv1.4, Kv2.1; 1:250 and Kv4.2; 1:200) and the cardiac calcium channel {alpha}1C subunit (1:200) were incubated with the membranes overnight at 4 °C. After primary antibody treatment, the membranes were washed with PBS–0.1% Tween-20 and incubated with a secondary antibody (horseradish peroxidase–conjugated goat anti-rat IgG) (Jackson ImmunoResearch). Immunoreactive protein bands were visualized on X-ray film (Kodak) using the enhanced chemiluminescence method (Pierce) and quantified by densitometry (Bio-Rad Molecular Imager). The density of each band was determined by averaging the pixel values within the region defined by cursors (Bio-Rad Quantity One software).

2.4 cAMP assay
Intracellular concentrations of cAMP were measured from control, forskolin (2 µM), IBMX (100 µM) and isoproterenol (1 µM) treated (5 min) myocytes. For this purpose the culture medium was aspirated and 0.1 N HCl added to the plates to cause cell lysis. Following centrifugation (1200xg, 10 min) the precipitates were solubilized in NaOH for determination of total protein by the Lowry method. The supernatant fractions were diluted and lyophilized to dryness. The dried residue was then re-suspended in assay buffer and cAMP levels measured using a commercial immunoassay kit (Sigma Chemical Co.).

2.5 Drugs and chemicals
Tetraethlyammonium (TEA), forskolin, isoproterenol, 4-aminopyridine, cAMP (Na+ salt) and 3-isobutyl-1-methylxanthine were purchased from Sigma (St. Louis, MO). TTX and PKI were purchased from CalBiochem (San Diego, CA). Antibodies to the Kv1.4, Kv4.2 and Kv2.1 {alpha} subunits and the N-terminal of the cardiac calcium channel {alpha}1C subunit were obtained from Alomone Labs LTD. (Jerusalem, Israel).


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1 Voltage-gated ion currents in aligned and flat neonatal rat myocytes
Fig. 1 shows an example of neonatal rat ventricular myocytes cultured on either aligned or non-aligned (random) collagen thin gels. Cells plated on non-aligned collagen gels display a flat, stellar-shaped morphology typical of cultured neonatal cardiac myocytes [13]. In contrast, cells plated on the aligned collagen substrate display an elongated, rod-like shape, with myofibrils distributed in parallel arrays [13]. Since the aligned myocytes express an in vivo-like morphology, we investigated whether voltage-gated ion channels would also be regulated differently in these cells.

The top panel of Fig. 2 displays outward, voltage-gated potassium currents measured in both aligned and flat neonatal myocytes. As reported for adult rat ventricular myocytes [18], potassium currents recorded in the neonatal myocytes consisted of two components: a transient outward current (Ito), which activated quickly and then inactivated within the first 100 ms of the pulse, and a sustained current (Isus), which was measured at the end of the pulse. Both potassium current components were completely eliminated when the myocytes were dialyzed with an internal solution containing 130 mM cesium (Figs. 4 and 5Go). In order to quantify the Ito component, the current measured at the end of the voltage pulse was subtracted from the peak Ito for each record, and the resulting current was then normalized to the cell capacity. The bottom panel of Fig. 2 displays the current vs. voltage relationship for Ito measured in both the aligned and flat myocytes. The peak current density of Itowas over 2-fold larger in the aligned myocytes (Fig. 2, Table 1).


Figure 2
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  		Fig. 2 Transient outward K+ currents are increased in aligned myocytes. Top panel: currents recorded during voltage steps, given in 10 mV increments, applied from a holding potential of –80 mV to potentials ranging from –20 to +50 mV. Bottom panel: average current vs. voltage relationship for Ito measured in a representative group of aligned (n = 10) and flat (n = 10) myoyctes and normalized to the cell membrane capacity.

 

Figure 4
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  		Fig. 4 Measurement of voltage-gated Na+ currents in aligned and flat myoyctes. Top panel: currents recorded during voltage steps, given in 10 mV increments, applied from a holding potential of –80 mV to potentials ranging from –50 to +20 mV. Bottom panel: mean current vs. voltage relationship for INa measured in a representative group of aligned (n = 6) and flat (n = 6) myoyctes and normalized to the cell membrane capacity.

 

Figure 5
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  		Fig. 5 L-type Ca2+ currents are increased in aligned myoyctes. Top panel: currents recorded during voltage steps, given in 10 mV increments, applied from a holding potential of –40 mV to potentials ranging from –30 to +10 mV. Bottom panel: average current vs. voltage relationship for ICa measured in a representative group of aligned (n = 9) and flat (n = 10) myoyctes and normalized to the cell membrane capacity.

 

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  		Table 1 Summary of voltage-gated current amplitudes in flat and aligned cardiac myocytes

 
Despite the difference in magnitude of the Ito, both cell phenotypes displayed a similar sensitivity to the potassium channel blocker 4-aminopyridine (2 mM) with reductions of 77±3% vs. 74±3% in the flat (n = 3) and aligned myocytes (n = 7).

One possible explanation for the enhanced Ito in the aligned myocytes could be that the activation or inactivation properties of the current are altered when the cells are plated on aligned substrates. Alignment of cells might cause a shift of the activation voltage to a more negative potential or cause inactivation to be shifted to a more positive potential. Fig. 3 (top panel) displays activation and steady-state inactivation curves for Ito measured in both the aligned and flat cells. Slight differences in voltage-dependence of activation and steady-state inactivation were recorded in the two cell phenotypes. For the aligned cells, relative to flat cells, the half-maximal voltage (V1/2) required for activation was shifted to a more negative potential (12±1 mV for aligned cells, 17±2 mV for flat cells), and the V1/2 of steady-state inactivation was shifted to a more positive potential (–32±1 mV for aligned cells, –36±1 mV for flat cells). However, these small changes in the activation and inactivation V1/2's can not account for the large change in the Ito density.


Figure 3
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  		Fig. 3 Properties and expression of voltage-gated K+ channels in aligned and flat myocytes. Top panel: activation and inactivation curves obtained in the myocytes. For activation, conductance was determined by dividing the peak current amplitude at each potential by the driving force for K+, (Vm–EK). The continuous lines represent the best fits of the Boltzmann equation, gK=gKmax{1+exp[–(Vm–V1/2)/k]}, where V1/2 is the half-maximal voltage required for activation and k gives the steepness of the voltage-dependence to the data points. Inactivation curves were obtained using a two-pulse protocol. Currents obtained at +30 mV were normalized and plotted as a function of the prepulse potential. Data were fit with the equation: IK=IK(max)/(1+exp[(Vm–V1/2)/k]), where V1/2 is the half-maximal voltage required for inactivation and k the slope. For the activation curves, each point represents the mean±S.E. obtained from 16 cells. For the inactivation curves, points represent the mean±S.E. from either 21 aligned or 14 flat cells. There was no statistically significant change in the k value for either activation or inactivation curves. Bottom panel: Kv Western blot analysis. Cardiac cell lysates were prepared from both flat (F) and aligned (A) myocytes and cellular proteins (50 mg per lane) separated by SDS–PAGE. PVDF membranes were probed with antibodies to the Kv1.4, Kv4.2 and Kv2.1 {alpha} subunits and protein bands visualized as described in the text. These bands were completely eliminated when the Abs were preincubated with the peptides against which the Abs were generated (not shown).

 
Voltage-gated K+ channels are composed of Kv {alpha} subunits which form the pore of the protein. Since the Kv4.2 {alpha} subunit (MW{approx}75 kDa) is responsible for the cardiac Ito [19,20], it was determined whether the expression of this subunit was altered by changes in cell morphology.

Despite the larger Ito density found in the aligned cells, there was no difference in the expression of the Kv4.2 subunit in the aligned and flat cells (results of three myocyte cultures) (Fig. 3, bottom panel). We also examined the Kv1.4 (MW{approx}97 kDa) and Kv2.1 (MW{approx}120–130 kDa) {alpha} subunits which underlie transient and delayed rectifier K+ currents, respectively. Once again, no difference in the protein density for these subunits could be detected between the flat and aligned mycoytes (Fig. 3, bottom panel).

Consistent with the Ito results, plating the neonatal myoctyes on aligned collagen membranes caused the current densities of both the sodium (INa) and L-type calcium (ICa) currents to increase.

Fig. 4 shows examples of INa measured in aligned and flat myocytes. Consistent with previous studies of rat ventricular myocytes [21], a high concentration of TTX was required to block INa in the aligned myocytes (20 µM caused 95% block). Although the same TTX-resistant INa could be measured in the flat myocytes, the peak INa density in the flat cells was significantly smaller (Fig. 4, Table 1). The peak ICa density was almost 2-fold larger in the aligned cells (Fig. 5, Table 1).

The increased size of ICa in the aligned cells was accompanied by an enhanced rate of current inactivation. Fitting the decay phase of ICa at 0 mV with a single exponential function gave time constants of 19±2 ms for the aligned cells, and 29±4 ms for the flat cells (P<0.05, n = 25).

Although not tested, this increased rate of decay of the current may have resulted from more Ca2+-induced inactivation in the aligned cells.

3.2 Protein kinase regulation of ICa in aligned and flat neonatal myocytes
Cardiac calcium channels are strongly regulated by β-adrenergic receptor agonists and other agents which stimulate cAMP-dependent protein kinase (protein kinase A) [22,17]. Application of 2 µM forskolin and 100 µM 3-isobutyl-1-methylxanthine (IBMX), to elevate cAMP levels, caused ICa in the flat myocytes to increase by over 100% (Fig. 6). Surprisingly, addition of these two agents to the aligned myocytes resulted in a significantly smaller increase in ICa (Fig. 6). The response of ICa in both the aligned (Fig. 6) and flat cells was eliminated during cellular dialysis with the protein kinase A 6-22 amide inhibitory peptide (PKI, 20 µM). In myocytes dialyzed with PKI, ICa increased by 3±6% (six aligned cells) and 6±5% (four flat cells) following application of forskolin/IBMX. In addition, the basal ICa, measured between the 1st and 5th min following breakthrough of the patch pipette (containing PKI) into the cells, decreased by 7±1% (n = 6) in the aligned cells and by 8±1% in the flat cells (n = 4). In contrast, the basal current increased by 4±2% (n = 4 aligned cells) in myocytes dialyzed with heat-inactivated PKI.


Figure 6
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  		Fig. 6 Regulation of L-type Ca2+ currents in neonatal and young adult ventricular myocytes. Top panel: currents recorded from flat neonatal and young adult myocytes during a voltage step applied to 0 mV in the presence and absence of 2 µM forskolin and 100 µM 3-isobutyl-1-methylxanthine (IBMX). Bottom left panel: currents recorded from aligned neonatal myocytes during a voltage step applied to 0 mV in the presence and absence of 2 µM forskolin and 100 µM 3-isobutyl-1-methylxanthine (IBMX). Cells were dialyzed with either control or protein kinase inhibitor (PKI)-containing internal solution. Bottom right panel: percent increase in ICa measured in the presence of forskolin and IBMX. Each bar represents the mean±S.E. increase obtained in the flat (n = 7), aligned (n = 8) and young adult (n = 6) myocytes.

 
In order to determine if the small change in calcium channel regulation in the aligned cells resulted specifically from changes in cell shape, ventricular myocytes were isolated from 4-week-old rats. These myocytes provided a useful model for studying ICa regulation since these cells display a rod-like shape when plated on laminin or collagen substrate, yet do not represent a fully developed adult myocyte. As with the aligned myocytes, the cells from the 4-week-old rats expressed a large ICa density (Table 1) with a relatively fast time constant of inactivation (18±2 ms, n = 8). However, unlike the aligned myocytes, ICa in these cells was strongly stimulated by forskolin and IBMX (Fig. 6).

One possible explanation for the smaller response of ICa in the aligned neonatal myocytes to forskolin and IBMX could be that the channels are not responsive to elevations in cAMP. In order to test this hypothesis, the effect of direct cellular dialysis with cAMP was determined. When compared with the non-dialyzed cells, no significant change in the size of ICa was measured when the aligned myocytes were exposed for 5–15 min to internal solution containing 100 µM cAMP (compare Figs. 5 and 7Go). In contrast, ICa in the flat cells increased from 7±1 to 12±1 pA/pF in the presence of this same cAMP solution (compare Figs. 5 and 7Go). Additionally, there was no statistical difference in the ICa density measured with cAMP in the flat (12±1 pA/pF, n = 10) and the aligned (16±2 pA/pF, n = 12) myocytes (P>0.05).


Figure 7
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  		Fig. 7 L-type Ca2+ currents in the aligned myocytes are not regulated by cAMP. Top panel: currents recorded during voltage steps, given in 10 mV increments, to potentials ranging from –30 to +10 mV during cellular dialysis with 100 µM cAMP. Bottom panel: average current vs. voltage relationship for ICa measured during cAMP dialysis in the aligned (n = 12) and flat (n = 12) myocytes and normalized to the cell membrane capacity.

 
The results obtained with the aligned myocytes might suggest that basal cAMP levels are elevated in these cells. If cAMP concentrations are high in the aligned cells under control conditions, this would explain the relative insensitivity of the calcium channels to forskolin/IBMX and cAMP dialysis. However, as shown in the top panel of Fig. 8, there was no difference in the basal cAMP concentrations measured in the flat and aligned cultures. In addition, cAMP levels increased by a similar extent in both the flat and aligned myocytes in the presence of forskolin (2 µM), IBMX (100 µM) and isoproterenol (1 µM) (Fig. 8).


Figure 8
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  		Fig. 8 cAMP concentrations and expression of the {alpha}1C subunit in aligned and flat myocytes. Top panel: cAMP concentrations measured under control conditions or in the presence of forskolin (For), IBMX or isoproterenol (Iso) in the flat and aligned cells. Each bar represents the mean±S.E. obtained from three to five cultures. Bottom panel: Calcium channel Western blot analysis. Cardiac membrane fractions were prepared from flat (F), aligned (A) and 4-week-old juvenile (J) myocytes and cellular proteins (70 mg per lane) separated by SDS–PAGE. PVDF membranes were probed with an antibody to the N terminus of the {alpha}1C subunit (Card-N) either in the absence or presence of the N1–46(S44A) GST-fusion protein (FP) [23].

 
In the final set of experiments, we tested the hypothesis that cardiac calcium channel {alpha}1C subunit is expressed at high levels in the aligned cells and that this elevated level of expression results in a decreased responsiveness of ICa to cAMP. The bottom panel of Fig. 8 displays a Western blot obtained using an antibody directed against the N-terminus of the {alpha}1C subunit (Card-N). As described previously [23,24], an immunoreactive band of approximately 210 kDa was identified using the anti-Card-N Ab. When compared with the flat myocytes, the density of this band was increased by 171±52% in the aligned cells (n = 5 cultures of aligned and flat cells). An additional minor band of approximately 160 kDa [23] was also present in the aligned cells. Both bands were completely eliminated in the presence of the N1–46(S44A) GST-fusion protein (FP) against which the anti-Card-N Ab was raised (Fig. 8) [23]. A band of approximately 240 kDa was identified in the cardiac myocytes obtained from the 4-week-old rats (Fig. 8).


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
4.1 Voltage-gated ion channels in flat and aligned myocytes
Interactions between the extracellular matrix and cardiac sarcolemmal membrane are important in determining the phenotype and function of the developing myocyte [8]. It was shown in the present study that neonatal rat ventricular myocytes plated on aligned collagen gels have increased densities of transient outward potassium (Ito), TTX-resistant sodium (INa) and L-type calcium (ICa) currents, when compared with cells plated on non-aligned flat collagen. Although the non-normalized current amplitudes of Ito and INa were not statistically different in the two cell morphologies (see Table 1), the magnitude of ICa was increased in the aligned cells despite a reduced cell capacity (Table 1). This augmentation of ICa in the aligned cells was associated with an increased expression of the calcium channel {alpha}1C subunit (Fig. 8).

When first plated onto collagen or laminin coated culture dishes, neonatal ventricular myocytes are spherical in appearance. However, within 24 h in culture the cells begin to spread and flatten out on the substrate, eventually taking on a stellate appearance [12,13] (Fig. 1). On days 2–7 in cell culture the ICa density has been reported to range from approximately 2 to 7 pA/pF when physiological concentrations of Ca2+ are present as the charge carrier [25,26]. Similar current densities can be measured in freshly isolated neonatal cells [27]. The value of 7±1 pA/pF, that we have determined for the flat cells on culture days 3–4, is consistent with these measurements. Developmental studies indicate that ICa densities increase slightly to 8–10 pA/pF in freshly isolated adult ventricular myocytes [25–27]. The current density of 13 pA/pF found in the aligned cells is therefore unusually high for a rat ventricular myocyte. While there have been no direct studies on the relationship of cardiac cell shape to Ca2+ channel expression, Matsuda et al. [11] found that acute changes in cell shape, induced through osmotic cell swelling and cell inflation, increase ICa by over 30% in rabbit myocytes. The mechanically induced increase in ICa is not mediated through a stimulation of protein kinase A, since dialysis of the cells with a protein kinase inhibitor peptide does not prevent this stimulation [11]. This finding parallels previous reports showing that changes in cell volume increase the activity of the slow, delayed rectifier K+ channel (IKs) [9].

Profound developmental increases in current amplitudes have been reported for cardiac outward potassium currents such as Ito. Xu et al. [28] measured Ito in ventricular cells obtained from postnatal rats on days 5 through 30 and from adult rats. In addition, changes in the expression of the potassium channel {alpha} subunit Kv4.2, which functions as the major contributor to Ito [19,20], were quantified. Ito was found to increase from 6 pA/pF in the 5-day-old neonatal animals to 20 pA/pF in the 8- to 12-week-old adult animals [28]. The increase in Ito was accompanied by an age-dependent increase in the Kv4.2 {alpha} subunit [28]. Qualitatively similar developmental increases in Ito density have been described by other investigators [29,30]. In general, this increase in Ito density is not accompanied by any significant changes in the voltage- or time-dependent properties of the currents [29,28], although the rate of recovery of Ito from steady-state inactivation has been reported to increase in the older animals [31]. This change in the inactivation recovery time may be related to a decreased Kv1.4 {alpha} subunit expression which occurs as the animals age [32,28]. In the present study, plating the neonatal myocytes on aligned collagen gels caused a 2-fold increase in the Ito density. The small difference noted between the aligned and flat cells in the voltage-dependence of activation and steady-state inactivation may have been indicative of changes in the ratio of the Kv4.2 and Kv1.4 subunits. However, using Western blot analysis we could find no evidence for differences in the expression of the Kv4.2, Kv1.4 and Kv2.1 {alpha} subunits in the flat and aligned myocytes.

4.2 Protein kinase A regulation of ICa
Since the aligned cells displayed an in vivo-like morphology, and since the ICa densities in these cells were closer to those from adult rats, we had expected to observe a large regulatory effect on ICa during stimulation of protein kinase A.

Surprisingly, the aligned cells responded with a relatively small change in ICa during application of forskolin and IBMX. In some cells, the currents increased by only 10–20%. Furthermore, there was no significant change in the amplitude of ICa when the aligned cells were dialyzed with 100 µM cAMP. Although we hypothesized that the large ICa density might result from an elevated level of cAMP in these cells, intracellular concentrations of cAMP were not different between the flat and aligned cells (Fig. 8). In addition, the basal ICa was reduced by a similar amount in the two cell morphologies during PKI dialysis. Since both the flat and aligned myocytes responded to forskolin with similar increases in cAMP production, our results indicate that ICa in the aligned cells is not responsive to elevations in cAMP. Thus although the aligned cells have high ICa densities, the regulatory properties of the channels in these cells are clearly different from those of myocytes isolated from 4-week-old animals.

Muth et al. [33] have reported that cardiac myocytes obtained from transgenic mice that over-express the L-type Ca2+ channel, display a reduced sensitivity to β-adrenergic/adenylate cyclase stimulation. In addition, clinical studies have demonstrated that the heart is less responsive to β-adrenergic agonists under conditions of serum hypercalcemia [34]. The increased expression of the calcium channel {alpha}1C subunit (Fig. 8), possibly by elevating intracellular Ca2+ levels, may produce a similar depression of the cAMP regulatory pathway in the aligned cells. However, this hypothesis is not consistent with the strong regulatory effect of forskolin/IBMX on ICa measured in the 4-week-old myocytes, that expressed levels of the {alpha}1C subunit comparable to that of the aligned cells (Fig. 8). The mechanism of this interaction between the {alpha}1C subunit, intracellular Ca2+, and β-adrenergic regulation will require further investigation.

Several studies have demonstrated that β-adrenergic receptor/adenylate cyclase stimulation is associated with a 100–120% increase in ICa in normal flat neonatal and adult rat myocytes [25–27].

However, β-adrenergic responsiveness of the myocytes is decreased under conditions that cause changes in myocyte morphology. For example, β-adrenergic-induced increases in ICa are diminished in adult rat myocytes isolated from hypertrophied and infarcted hearts [35,36]. Transgenic mice over-expressing the G protein, Gq, display cardiac hypertrophy with a ventricular ICa that responds poorly to the β-adrenergic agonist isoproterenol and to forskolin [37]. In addition, a recent study using cat atrial myocytes has shown that the β-adrenergic responsiveness of ICa is different in cells plated on glass and laminin [38]. This difference is eliminated when cells are plated in the presence of {alpha}β1-integrin antibody [38]. These results imply that interactions between cell membrane integrins and the surrounding extracellular matrix are critical for maintaining normal cell signaling. Future experiments manipulating the expression of {alpha} and β intergrins, to induce morphological changes in the aligned myocytes, will provide more information on the relationship between cardiac cell shape and ion channel regulation.

Time for primary review 23 days.


   Acknowledgements
 
This work was supported by US Public Health Service award HL-45789, grants from the Mid-Atlantic Consortium of the American Heart Association and the Research Development Fund of the University of South Carolina, School of Medicine. The authors thank Ms Kathryn J. Long for preparing the neonatal myocytes and Ms Jining Zhang for her assistance with the Western blot experiments.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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