Cardiovascular Research

Cardiovascular Research 1999 41(1):157-165; doi:10.1016/S0008-6363(98)00157-6
© 1999 by European Society of Cardiology

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Copyright © 1999, European Society of Cardiology

Paracrine hypertrophic factors from cardiac non-myocyte cells downregulate the transient outward current density and Kv4.2 K⁺ channel expression in cultured rat cardiomyocytes

Weinong Guo^a,b,*, Kaichiro Kamiya^a, Kenji Yasui^a, Itsuo Kodama^b and Junji Toyama^a

^aDepartment of Circulation, Research Institute of Environmental Medicine, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan
^bDepartment of Humoral Regulation, Division of Regulation of Organ Function, Research Institute of Environmental Medicine, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan

^* Corresponding author. Present address: Department of Molecular Biology and Pharmacology, Washington University School of Medicine, Box 8103, 660 South Euclid Avenue, St. Louis, MO 63110, USA. Fax: +314-362-7058.

Received 28 January 1998; accepted 6 May 1998

	Abstract

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
Objectives: Cardiac hypertrophy is characterized by a prolongation of action potential duration (APD) and a reduction of outward K⁺ currents, primarily the transient outward current (I_to). Since the interaction between cardiac non-myocyte cells (NMCs) and cardiomyocytes (MCs) plays a critical role during the process of myocardial hypertrophy, in the present study, we investigated the effects of NMCs on cell growth and K⁺ channel expression in cultured newborn rat ventricular cells. Methods: Single MCs were isolated from day-old Wistar rat ventricles and cultured for a period of five days. The effects of NMCs were examined by MC–NMC co-culture or incubating pure MCs in NMC-conditioned growth medium (NCGM). Whole-cell voltage–clamp recording and Western blot analysis using a polyclonal antibody against rat Kv4.2 channel protein were performed. Results: A marked increase in surface area and total cell protein concentration of MCs was observed in the MC–NMC co-culture. In the pure MC culture, this hypertrophic effect could be mimicked by a 72-h addition of NCGM, with a significant prolongation of APD₂₅ (APD at 25% repolarization) and a 42% decrease in I_to density (at +30 mV). The rates of inactivation and recovery from inactivation of I_to were unchanged. In the NCGM-treated MC culture, Western blots of MC proteins also showed a 36% reduction of the Kv4.2 K⁺ channel protein level. In addition, the NCGM-induced MC hypertrophy was partially inhibited by anti-insulin-like growth factor-1 (IGF-1) antibody, while it revealed no effects on I_to density and Kv4.2 channel expression. Conclusions: These findings first demonstrate that some paracrine hypertrophic factors released from cardiac NMCs, although unidentified, downregulate cardiac K⁺ channel expression.

KEYWORDS Hypertrophy; Cardiomyocyte; Cardiac non-myocyte cell; Transient outward current; Shal

	1 Introduction

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
Myocyte hypertrophy is known to be a process of cardiac adaptation when subjected to a chronic increase in haemodynamic load, and longstanding hypertrophy can often result in the subsequent onset of overt heart failure (for review, see [1]). A number of experimental studies have provided evidence concerning the reduction of voltage-gated K⁺ channel currents during cardiac remodeling that leads to a prolongation of action potential duration (APD) (for reviews, see [2, 3]). Although the discrepancy still remains, depending on channel phenotypes, it has been consistently demonstrated that there is a dramatic decrease in transient outward current (I_to) in hypertrophied ventricular myocytes, such as genetic hypertension [4]and hypertrophy secondary to pressure overload [5]or to hormonal intervention [6]. Such channel regulation contributes to the electrical abnormality observed in hypertrophied heart, while its underlying mechanism is not clearly understood.

The heart functions as a syncytium of cardiomyocytes (MCs) and the surrounding non-myocyte cells (NMCs), which consist of fibroblasts, endothelial cells, smooth muscle cells and macrophages. Although cardiac MCs make up most of the adult myocardial mass, they comprise less than 30% of the total cell number in the heart, the rest being composed of NMCs [7]. During myocardial hypertrophy in vivo, in addition to MC enlargement, proliferation of NMCs and progressive interstitial and perivascular fibrosis are considered to be associated with abnormal cardiac function [8, 9]. There is a hypothesis that the interaction between cardiac NMCs and MCs plays a critical role not only in MC hypertrophy but also in ion-channel regulation. In the present study, we investigated the effects of NMCs on cell growth and K⁺ channel expression in cultured newborn rat ventricular MCs. Our observations suggest that paracrine factors secreted from cardiac NMCs stimulate MC hypertrophy and downregulate the I_to density and Kv4.2 K⁺ channel subunit, a key component of rat ventricular I_to [10–12]. This is particularly important for understanding the cellular mechanisms involved in cardiac ion-channel remodeling.

	2 Methods

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
2.1 Isolation and culture of cardiomyocytes
Single newborn ventricular MCs were isolated from day-old Wistar rats. All of the procedures conform 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). Briefly, hearts were rapidly removed via thoracotomy after a subcutaneous injection of sodium pentobarbital. Both ventricles were cut into 1- to 2-mm cubes and dissociated at 37°C for 10 min in Dulbecco's phosphate-buffered saline (PBS) solution containing 0.1 mg/ml collagenase (Yakult, Japan). Cell suspensions obtained in this way were pelleted by centrifugation at 800xg for 5 min. Cells were then resuspended in normal growth medium at 10⁵/ml. After 30-min preplating twice to purify the MC population, the medium supernatant containing primarily unattached MCs (>95%) was seeded onto 5x5 mm coverslips (Matsunami Glass, Japan) in multiwell 35-mm culture dishes (Falcon, USA). The normal growth medium used was a bicarbonate-buffered Eagle's MEM (Nissui Pharmaceutical, Japan) supplemented with 2% heat-inactivated newborn calf serum (Gibco, USA) plus 0.3 mg/ml glutamine, and was renewed daily. These control pure MC cultures were maintained at 37°C for five days in a humidified incubator (Sanyo, Japan) containing 5% CO₂ and 95% air.

2.2 Development of cardiomyocytes in vitro
To investigate the effects of NMCs, MC–NMC co-cultures and pure MC cultures treated with NMC-conditioned growth medium (NCGM) were established. Initial preplating was omitted in order to develop a five-day MC–NMC co-culture. In a prior study by us [13], it was shown that NMCs accounted for 25–35% of the total number of cells in this co-culture system. Moreover, from day three, pure MC cultures were incubated in NCGM for a period of 72 h. Cardiac NMCs were separated from day-old Wistar rat ventricles by the differential plating protocol described above. The NMCs obtained from two preplatings were washed with PBS, resuspended in normal growth medium and then incubated for two weeks. These cultured NMCs always reached confluence within one week (see Fig. 1). The growth medium was renewed every other day during the last seven days, and the medium supernatant was collected and stored at –20°C before use. NCGM contained 80% medium supernatant derived from the NMC cultures and was applied to the pure MC cultures with daily exchange. In addition, in one group of NCGM-treated MC cultures, a monoclonal antibody (Austral Biologicals, USA) against insulin-like growth factor-1 (IGF-1) was added to the growth medium at a concentration of 10 µg/ml for 72 h.

View larger version (37K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 NMCs induce enlargement of cultured cardiomyocytes. Single ventricular MCs were isolated from day-old Wistar rats and incubated for a period of five days under various growth conditions. Phase-contrast images of cultured cells are shown for a control pure MC culture, a two-week confluent NMC culture, a MC–NMC co-culture (M and N indicate MCs and NMCs, respectively), a MC culture treated with NCGM and a MC culture treated with NCGM plus monoclonal anti-IGF-1 antibody (10 µg/ml).

 
2.3 Measurement of myocyte growth
Cell size of cultured MCs was quantified by surface area and total cell protein concentration. MC surface area was determined by planimetry of cells on calibrated photomicrographs [14]. Briefly, fields were randomly selected from each of three or more dishes for photography. By using NIH Image 1.41 software to measure the area of MCs on the calibrated photomicrographs (taken at x400 and enlarged to 10x7 cm), the total surface area of each MC, in µm², was determined by doubling the calculated area to account for the surface in contact with the dish. Usually, we measured 30–50 MCs for each datum point.

To assess the total cell protein concentration, cell number was first calculated by counting, at x400, all attached MCs in 50 randomly selected fields that sampled the entire dish surface. The mean total cells/mm² was multiplied by 800 mm² to give the total number of MCs per dish (the value of 800 mm², being the surface area of a 35-mm culture dish, was supplied by Falcon). After counting the cell numbers, dishes were rapidly washed three times with PBS to remove serum, and MCs were dissolved in 0.1% sodium dodecyl sulfate (SDS, Katayama Chemical, Japan). Protein in each dish was measured in triplicate with the dye-binding assay [15]using a UV-1200 spectrophotometer (Shimadzu, Japan) with bovine serum albumin (Bio-Rad, USA) as the standard. At least nine–ten dishes were measured for each datum point and the protein content was divided by the total MC number per dish to give the cell's protein concentration in pg/cell.

2.4 Electrophysiological recordings
At day six of cell culture, coverslips holding the cultured MCs were transferred to a recording chamber mounted on an inverted microscope (Nikon, Japan). MCs were first perfused with Tyrode bath solution containing, in mM, NaCl 146.9, KCl 5.4, CaCl₂ 1.8, MgCl₂ 0.5, NaH₂PO₄ 0.33, HEPES 5.0 and glucose 5.0 (pH was adjusted to 7.4 using NaOH), for at least 30 min. Whole-cell recordings were then carried out at room temperature (22–25°C) using an L/M EPC-7 (D-6100 Darmstadt-13/w, Germany) or an Axon-200B (Axon Instruments, USA) patch–clamp system. Pipettes were pulled from borosilicate glass and, after fire polishing, had a resistance of 3–5 M when filled with pipette solution containing, in mM, KCl 140, NaH₂PO₄ 10, CaCl₂ 0.2, MgATP 5.0, EGTA 10 and HEPES 10 (pH was adjusted to 7.2 using KOH). The liquid junctional potential between the pipette and bath solutions was always corrected before the formation of a gigaohm seal. After breaking the membrane by applying a slow suction to the pipette, membrane capacitance (C_m) was calculated as the area under capacitive transient divided by the amplitude of an applied test pulse (5 mV) from a holding potential (HP) of –80 mV. The series resistance was electrically compensated by 70–80%. Action potentials of cultured MCs were first recorded in the current–clamp mode in Tyrode bath solution. They were evoked by 3- to 5-ms suprathreshold current pulses applied at 1.0 Hz and measurements were made after a steady-state was reached for 10 min. Thereafter, the bath solution was replace by test solution for recording K⁺ currents. Test solution was formed by adding 10 µM Tetrodotoxin (TTX) combined with 3 µM nisoldipine to Tyrode solution. All recordings of current were digitized at 2 KHz for on-line storage in a personal computer and were subsequently analyzed using PClamp 6.0 software (Axon Instruments). The density of measured current was calculated by normalization to C_m.

2.5 Polyclonal antibody against the Kv4.2 K⁺ channel subunit
Polyclonal antisera against Kv4.2 were generated, as previously reported [16], by immunizing rabbits with the recombinant fusion peptide that contained 19 amino acids (CLEKTTNHEFVDEQVFEES) corresponding to residues 484 to 502 in the C terminus of rat Kv4.2. The anti-Kv4.2 antibody was purified by affinity chromatography on Protein G–Sepharose columns.

2.6 Western blotting
To perform Western blot analysis, after a rapid rinse with PBS, MCs were scraped from culture dishes, lysed with boiling electrophoresis sample buffer (125 mM Tris–HCl, pH=6.8, 2% SDS, 5% glycerol, 0.003% bromophenol blue and 1% β-mercaptoethanol) and exposed to a brief sonication to reduce viscosity. A fixed amount of myocyte protein (50 µg) from each sample was fractionated by 12.5% SDS–polyacrylamide gel electrophoresis (SDS–PAGE) and transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, USA). To prevent non-specific reactions taking place, PVDF membrane strips were incubated in blocking buffer (3% ovalbumin and 0.02% NaN₃ in PBS) at 37°C for 1 h. For identification of the Kv4.2 K⁺ channel -subunit and -actin (a control protein to facilitate comparisons of samples), membrane strips were first exposed to primary antibody solution (anti-Kv4.2, 20 µg/ml or rabbit anti-actin, Sigma, St. Louis, MO, USA; 1:100, v/v; both diluted in the blocking buffer) at room temperature overnight and then incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG [Sigma, 1:500(v/v)] at 37°C for 30 min. Strips were rinsed three times in wash buffer containing 0.1% Tween 20 in PBS, and bound antibodies were finally detected using the enhanced chemiluminescent reagent (Amersham, UK). Films of Western blots were scanned into a Bioimage Analyzer (Hewlett Packard, USA). The densitometric value for each Kv4.2 channel protein band was quantified using NIH Image 1.41 software and then calculated as the ratio of Kv4.2 K⁺ channel -subunit to actin. Finally, these values for each sample were normalized to those determined for the bands of control samples on the same gel.

2.7 Statistics
All of the data are expressed as the mean±SD. Differences between mean values were assessed using Student's unpaired t-test for two groups, or by analysis of variance (ANOVA) followed by the Student–Newman–Keuls tests for comparisons of more than two groups. In all cases, P<0.05 was considered to be statistically significant.

	3 Results

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
3.1 NMCs stimulate MC hypertrophy
As shown in Fig. 1, when MCs were co-cultured with NMCs, an apparent enlargement of MCs was observed. NMC–MC co-culture induced a 93% increase in the MC surface area (P<0.05, Table 1). Interestingly, in the pure MC cultures, 80% replacement of growth medium with NCGM also induced 64 and 25% increases in the MC surface area and total cell protein concentration, respectively, at around 72 h of incubation (P<0.05). This NCGM-dependent hypertrophic effect could be partially inhibited by the addition of monoclonal anti-IGF-1 antibody (38 and 14% increases in cell size and cell protein concentration as compared with control; see Fig. 1Table 1).

View this table: [in this window] [in a new window]   Table 1 Cardiac non-myocyte cells (NMCs) induce myocyte hypertrophy in cultured newborn rat ventricular cells

 
3.2 Changes in action potentials
The similar MC hypertrophic effects between NCGM and NMC–MC co-cultures prompted us to further examine the characteristics of action potentials in the control pure MC cultures and the NCGM-treated MC cultures. In a whole-cell current–clamp mode, action potentials were evoked by 3- to 5-ms suprathreshold current pulses applied at 1.0 Hz. Compared with control MCs (Fig. 2A), action potentials recorded from the NCGM-treated MCs had a smaller initial repolarization and a pronounced plateau phase, resulting in 38 and 126% increases in the action potential overshoot and duration APD₂₅ (APD at 25% repolarization), respectively (P<0.05, Fig. 2B–C). On the other hand, there were no significant differences in resting potential (–66±5.7 mV, n=8 in control versus –68±6.2 mV, n=12 in NCGM-treated) and APD₈₀ (193±22 versus 208±34 ms) between these two culture preparations. In contrast to the inhibitory action of anti-IGF-1 in the NCGM-induced MC hypertrophy, it had no effect on the changes in action potentials (n=10, Fig. 2).

View larger version (21K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 Prolongation of action potential duration by treatment with NCGM. (A) Representative action potentials recorded from three pure MC cultures in a whole-cell current–clamp mode. Action potentials were evoked by 3- to 5-ms suprathreshold current pulses applied at 1.0 Hz. Measurements were made after a steady-state was reached for 10 min. Dotted lines indicate a zero voltage level. Comparison of action potential overshoot and action potential duration at 25% repolarization is illustrated in (B) and (C), respectively. Data were obtained from 8–12 MCs for each group (open bars: control pure MC cultures, n=8; hatched bars: NCGM-treated MC cultures, n=12; crossed bars: MC cultures treated with NCGM plus anti-IGF-1 antibody, n=10). #P<0.05 was considered to be significantly different from the control pure MC cultures.

 
3.3 Changes in I_to
I_to is a predominant repolarizing K⁺ current and determines the early phase of repolarization in rat heart action potential [17]. Fig. 3A illustrates a family of I_to currents that was evoked by various 300-ms depolarizing steps from a HP of –60 mV. I_to, characterized by rapid inactivation, could be detected in the majority of cultured MCs. Exposure to 4-aminopyridine (4-AP, 2 mM) preferentially blocked the transient component of outward currents by 90%, without any remarkable suppression of the sustained current at the end of the pulse (I_SUS). The amplitude of I_to was thus calculated as the difference in current between the peak current and I_SUS. As represented in Fig. 3B, I_to density (at +30 mV) in the NCGM-treated MC cultures (n=14) was 42% lower than that in the control MC cultures (n=10, P<0.05). Normalization of the current–voltage relationship (I/I_max) did not reveal any significant shift in the voltage-dependence of activation (data not shown). The anti-IGF-1 antibody had no effects on the NCGM-induced reduction of I_to density (38% decrease compared with control, n=9). In addition, comparative analysis of I_SUS in the MC cultures demonstrated that neither the treatment of NCGM nor the application of anti-IGF-1 antibody affected its current density (Fig. 3C).

View larger version (29K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 Reduction of cardiac Ito by treatment with NCGM. (A) Typical whole-cell Ito recordings from three pure MC cultures are demonstrated. From a HP of –60 mV, Ito was evoked by various depolarizing steps (from –10 to +40 mV, with an increment of 10 mV) for 300 ms at a cycle length of 10 s. Dotted lines indicate a zero current level. (B) and (C) show the current densities of Ito and ISUS (at +30 mV), respectively. Ito density was measured as the normalized difference current between the transient peak and sustained currents, while ISUS was estimated as the steady-state component at the end of a 300-ms voltage pulse. Data were obtained from four control pure MC cultures (open bars, n=10), three NCGM-treated MC cultures (hatched bars, n=14) and three MC cultures treated with NCGM plus anti-IGF-1-antibody (crossed bars, n=9). #P<0.05 was considered to be significantly different from the control cultures.

 
At room temperature, the time course of the first 150 ms of I_to decay at test potentials >0 mV could be precisely fitted by a single exponential equation: I_(t)=a* exp(–t/)+b, where is the time constant of current inactivation and b is a constant (Fig. 4A). As shown in Fig. 4B, similar voltage-dependences of I_to decay and identical -values were obtained among the control MC cultures (n=10), the NCGM-treated MC cultures (n=14) and the MC cultures treated with NCGM plus anti-IGF-1 (n=9). In addition, a standard double-pulse protocol was applied to study the recovery kinetics of I_to from inactivation (Fig. 4C). Two 150-ms pulses, each to +30 mV with varying interpulse intervals, were applied at 0.1 Hz from a HP of –60 mV. The magnitude of I_to elicited by the second pulse was normalized to the maximum current value (interpulse interval at 200 ms) and plotted as a function of recovery time (Fig. 4D). The recovery of I_to among the cultured cells incubated under these three growth conditions (n=5, respectively) could be fitted by a similar single-exponential function with a time constant of 41.5 ms.

View larger version (19K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4 Lack of effect of NCGM on the inactivation properties of Ito in cultured cardiomyocytes. (A) Illustration of the single exponential fit (solid lines) of the first 150 ms of Ito decay recorded from a control myocyte at test potentials from 0 to +40 mV, with an increment of 10 mV. The conclusive data obtained from four control pure MC cultures (circles, n=10), three NCGM-treated MC cultures (squares, n=14) and three MC cultures treated with NCGM plus anti-IGF-1-antibody (triangles, n=9) are shown in (B). A standard double-pulse protocol was applied at 0.1 Hz to examine the recovery kinetics of Ito from inactivation by using two 150-ms pulses at +30 mV, with varying interpulse intervals. (C) A typical example of Ito recovery measured from a control myocyte (arrow indicates the zero current level). Data were normalized to maximum current value (interpulse interval at 200 ms) and plotted as a function of recovery time (D). Results were obtained from five experiments for each datum point. Data under all of the three culture conditions fall on a similar curve and the solid line is a single exponential fit to the data.

 
3.4 Changes in the Kv4.2 K⁺ channel subunit
Western blotting was conducted on fixed amounts of MC proteins (50 µg) after fractionation on SDS–PAGE and transfer to PVDF membranes. Typical immunoblots obtained using the polyclonal antibody against rat Kv4.2 are presented in Fig. 5A. In the control sample, intense labeling of a single band at 74 kDa was detected by the anti-Kv4.2 antibody (lane a). The specificity of the antibody was confirmed by the observation that labeling of the 74-kDa band was blocked by preincubating the antibody with 50 µg of Kv4.2 fusion peptide against which the antibody was generated (lane d). The molecular mass (74 kDa) of this band was also similar to that of the Kv4.2 channel protein identified in newborn and adult rat ventricles [10, 18]. Weak labeling at 74 kDa was found in the NCGM-treated MC cultures, irrespective of the presence (lane c) or absence (lane b) of anti-IGF-1 antibody. To facilitate the comparisons between blots and among samples, each densitometric value of the Kv4.2 channel protein band was calculated as its ratio to the band of actin detected at 42 kDa, and then normalized to that determined for the control samples on the same gel. Treatment of pure MC cultures with NCGM for 72 h produced a 36% decrease in the mean Kv4.2 immunoreactive protein level (n=3, P<0.05), and this reduction was not affected by anti-IGF-1 (Fig. 5B).

View larger version (34K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 5 Western blot analysis of the Kv4.2 K+ channel subunit in cultured cardiomyocytes. Cell proteins (50 µg) obtained from each pure MC culture were fractionated on SDS–PAGE, transferred to PVDF membranes and immunoblotted with the polyclonal antibody against rat Kv4.2. Intense labeling of a single band at 74 kDa was detected by the anti-Kv4.2 antibody. In (A) (upper panel) representative immunoblots of myocyte proteins (50 µg) prepared from a control pure MC culture (lane a), a NCGM-treated MC culture (lane b) and a MC culture treated with NCGM plus anti-IGF-1-antibody (lane c) are shown. Lane d shows that the specific band detected by the antibody was eliminated by preincubating the antibody with 50 µg of Kv4.2 fusion peptide against which the antibody was generated. In the bottom panel, Western blots of -actin from the same samples are shown. Comparison of Kv4.2 K+ channel protein expression is illustrated in B. Films of Western blots were scanned into a Bioimage Analyzer (Hewlett Packard, USA). The densitometric value for each Kv4.2 channel protein band was quantified using NIH Image 1.41 software, calculated as its ratio to actin and, finally, normalized to those determined for the bands of control samples on the same gel. Data were derived from at least three independent experiments for each group (open bars: control pure MC cultures; hatched bars: NCGM-treated MC cultures; crossed bars: MC cultures treated with NCGM plus anti-IGF-1 antibody). #P<0.05 was considered to be significantly different from the control cultures.

 

	4 Discussion

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
This study clearly demonstrates the involvement of cardiac NMCs in the regulation of voltage-gated K⁺ channel expression during MC hypertrophy. It provides novel evidence about the importance of paracrine hypertrophic factors released from NMCs in cardiac ion-channel remodeling.

Cell–cell interaction between cardiac NMCs and MCs has been studied by a number of investigators using the NMC–MC co-culture system [19–22]. Simpson and Savion [19]showed that NMCs modified the differentiation properties of MCs. Long et al. [20]reported the secretion of a MC hypertrophic factor from NMCs that had a molecular mass of 45–50 kDa. And most recently, endothelin-1 secreted from cardiac NMCs has been identified as a factor related to MC hypertrophy [21, 22]. In the present study, an increase in both the MC surface area and the total cell protein concentration was observed in the NCGM-treated pure MC cultures. This MC hypertrophic effect mimicked that of the NMC–MC co-culture and indicates that some soluble paracrine factors produced from cardiac NMCs regulate MC hypertrophy. Although we separated NMCs from MCs using the conventional preplating protocol [19, 21]and not by the discontinuous gradient method that strictly isolates fibroblasts [22], the origin of such unidentified factors might be fibroblasts, since cardiac NMCs are reported to consist primarily of fibroblasts and a small amount of other cell types [7].

IGF-1, a 70-amino acid basic peptide, has been known to have diverse functions that mediate broad physiological processes, such as cell growth and differentiation, induction of cell proliferation and extracellular matrix production of vascular smooth muscle cells which contributes to angiogenesis [23–25]. Cardiac MCs also have been demonstrated to be targets for IGF-1 and it serves as an essential factor for cardiac development and hypertrophy [23, 26]. For example, Ito et al. [26]reported that the addition of exogenous IGF-1 to cultured newborn rat heart cells induced MC hypertrophy with increased expression of some muscle-specific genes. One of the major findings of the present study is that an antibody against IGF-1 partially inhibited the trophic effect of NCGM. This result strongly indicates that IGF-1 is one of the components in NCGM that mediates MC hypertrophy. Nevertheless, it should also be noted that the NCGM-dependent MC hypertrophy could not be completely blocked by anti-IGF-1 antibody, raising the possible involvement of other paracrine hypertrophic factors from NMCs.

The electrical activity of the heart underlies the mechanics of the cardiac pump. The duration of cardiac action potential, which is determined primarily by the voltage-gated K⁺ channel currents, is critical to myocardial pump performance and membrane excitability. Numerous experimental studies have shown the reduction of voltage-gated K⁺ currents in hypertrophied MCs resulting in a prolongation of APD (for reviews, see [2, 3]). Since NMCs regulate MC hypertrophy and the interaction between NMCs and MCs is considered to play a critical role in the process of cardiac remodeling, it should be of great interest to investigate the role of NMCs in the regulation of cardiac K⁺ channel expression. Up until now, Dourado and Dryer [27]were the only people who reported that co-culture with fibroblasts did not allow normal expression of A-current in embryonic chick parasympathetic neurones. By using the pure MC cultures, we studied the effects of NMCs on I_to channel activity. Incubation with NCGM induced a significant decrease in I_to density, without causing any changes in the kinetics of whole-cell current decay and recovery from inactivation. This reduced I_to may contribute to the increase in action potential overshoot and the prolongation of early repolarization (see Fig. 2). It is qualitatively similar to those in remodeling heart (see reviews [2, 3]). These results suggest that some paracrine factors from cardiac NMCs not only initiate the MC hypertrophic response but also suppress I_to. On the other hand, the effects of NMCs on cardiac K⁺ channel activity appear to depend on channel phenotypes. In contrast to I_to, treatment with NCGM did not cause any changes in I_SUS density. 4-AP-insensitive I_SUS, also referred as I_K, is another major K⁺ current in rat heart cells, distinct from I_to [17]. It determines the slow phase of action potential repolarization back to the resting membrane potential [17]. This might explain our findings that identical values of APD₈₀ were obtained between the control and the NCGM-treated MC cultures.

In adult rat heart, the Kv4.2 subunit, one of the members of the shal subfamily, is the most abundant channel protein, compared with other voltage-gated K⁺ channel subunits [10]. Kv4.2 transcript is expressed transmurally in rat ventricle, which is consistent with the distribution of I_to [11]. In addition, the Kv4.2 current expressed in mammalian cell displays flecainide sensitivity similar to native rat I_to [12]. These evidences support the hypothesis that Kv4.2 is the key molecular component of rat I_to. With this in mind, we performed Western blot analysis using a polyclonal antibody against the rat Kv4.2 channel. In a parallel manner to the decrease in I_to, a substantial reduction of the 74-kDa Kv4.2-immunoreactive protein level was also detected in the NCGM-treated MC cultures. These observations are in agreement with the conclusion that the paracrine trophic factors produced from NMCs downregulate the cardiac Kv4.2 K⁺ channel protein expression, which may underlie a decrease in sarcolemmal I_to channel density. Myocardial hypertrophy is always characterized by selective up- and downregulation of specific genes (see review [1]). Further study is required to elucidate the NMC-dependent transcriptional control of cardiac K⁺ channel expression that is presumably the molecular mechanism of our findings.

In adult rat myocardium, there is strong discriminatory evidence favoring Kv4.2 over Kv1.4 as an important molecular candidate of I_to [10–12]. However, at the early neonatal stage, the Kv1.4 channel is abundantly expressed at both the mRNA and protein levels [18], and the predominant K⁺ channel mRNA species switches from Kv1.4 to Kv4.2 during postnatal development [18, 28]. These results may indicate the unique participation of the Kv1.4 subunit in forming I_to in newborn rat cardiac MCs. This possibility seems unlikely since we observed that I_to in cultured newborn rat ventricular cells recovered from inactivation rapidly, with a time constant (=41.5 ms) comparable to that of the Kv4.2 current expressed in mouse L-cell lines (<100 ms, [12]). It is totally different from the slow recovery of either homotetrameric (=3.2 s) or heterotetrameric (=0.7–1.3 s) Kv1.4 channels [29].

Finally, the observation that treatment of pure MC cultures with NCGM revealed a downregulation of both I_to density and the Kv4.2 K⁺ channel protein level raises a question: what factors in NCGM trigger such changes? Considering the inhibitory effects of anti-IGF-1 antibody on NCGM-induced MC hypertrophy, it is plausible that IGF-1 may underlie the reduction in K⁺ channel expression. Strikingly, neither the NCGM-induced decrease in I_to density nor the suppression of the Kv4.2 channel was affected by anti-IGF-1. This implies that (1) some additional unidentified paracrine factors secreted from NMCs, other than IGF-1, contribute to the regulation of K⁺ channels during MC hypertrophy and (2) the NMC-dependent processes of MC hypertrophy and ion-channel remodeling are regulated, at least partially, via independent mechanisms. Further detailed analysis will be undertaken to clarify the ion-channel regulating factors that are responsible in NCGMs.

Time for primary review 22 days.

	Acknowledgements

 
We thank Dr. Jeanne M. Nerbonne (Washington University, USA) for critical advise on generating the anti-Kv4.2 antibody and Mrs. Mayumi Hojo for technical assistance in the experiments. This work was supported in part by a Grant-in-Aid for Scientific Research (A) from the Japanese Ministry of Science, Education, Sports and Culture (No. 07407073) and a grant from the Study Group of Molecular Cardiology of the Japanese Heart Foundation.

	References

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 

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