On the fate of skeletal myoblasts in a cardiac environment: down-regulation of voltage-gated ion channels

Abstract

We have analysed the voltage-gated ion channels and fusion competence of skeletal muscle myoblasts labelled with green fluorescent protein (GFP) and the membrane dye PKH transplanted into the infarcted myocardium of syngenic rats. After cell transplantation the animals were killed and GFP⁺−PKH⁺ myoblasts enzymatically isolated for subsequent studies of ionic currents through voltage-gated sodium, calcium and potassium channels. A down-regulation of all three types of ion channels after engraftment was observed. The fraction of cells with calcium (68%) and sodium channels (65%) declined to zero within 24 h and 1 week, respectively. Down-regulation of potassium currents (90% in control) occurred within 2 weeks to about 30%. Before injection myoblasts expressed predominantly transient outward potassium channels whereas after isolation from the myocardium exclusively rapid delayed rectifier channels. The currents recovered completely between 1 and 6 weeks under cell culture conditions. The down-regulation of ion channels and changes in potassium current kinetics suggest that the environment provided by infarcted myocardium affects expression of voltage-gated ion channels of skeletal myoblasts.

Compared to skeletal muscle the myocardium has a very limited potential to regenerate (Soonpaa & Field, 1998). After an infarction the muscle is transformed into a non-contractile fibrous scar. The remodelling process leads to expansion of the initial infarct area and to dilatation of the ventricular lumen (Pfeffer et al. 1991). The division of surviving cells in the infarct border zone (Beltrami et al. 2001) or recently identified stem cell populations (Jackson et al. 2001; Orlic et al. 2001, 2002; Quaini et al. 2002; Laflamme et al. 2002) may, to a certain extent, contribute new cardiac myocytes.

However, division of adult cardiomyocytes and de novo differentiation of cardiac myocytes from progenitor cells are clearly insufficient to compensate the functional damage. Cellular cardiomyoplasty has therefore been proposed as a new therapeutic option that may potentially stop or even reverse the process of postinfarction remodelling (Murry et al. 2002; Reinecke & Murry, 2003; Van Den Bos & Taylor, 2003; Menasché, 2003). Different cell types including myocardial cells, haematopoetic progenitor cells and skeletal muscle myoblasts have been shown to promote tissue regeneration in a number of chronic myocardial injury models (Koh et al. 1993; Taylor et al. 1998; Li et al. 1999; Hutcheson et al. 2000; Kocher et al. 2001).

Autologous skeletal myoblasts transplanted into the infarcted myocardium improve myocardial performance in vitro and in vivo. After 1–3 days these cells differentiate into myotubes and acquire the features of maturing myofibres that retain the skeletal muscle phenotype (Murry et al. 1996). Apparently, these fibres are not electrically coupled to surrounding myocardial cells (Murry et al. 2002; Leobon et al. 2003). Recent studies have shown that grafted myoblasts differentiate into hyperexcitable myotubes with a contractile activity fully independent of neighbouring cardiomyocytes (Leobon et al. 2003). It remains currently unclear how these fibres contribute to cardiac contractility (Menasché, 2003).

In the present study we analyse the current density and kinetic features of voltage-gated sodium, calcium and potassium channels in rat myoblasts before and after transplantation into the infarcted rat myocardium. A down-regulation of voltage-gated sodium, calcium and potassium channels progressing during engraftment was observed. However, our data also show that these cells can re-enter the cell cycle, retain their fusion competence and display a recovery of ion channels within 1–6 weeks under cell culture conditions.

Methods

The animal experiments were approved and performed according to the guidelines of the Animal Care Commission of the University of Innsbruck and by the Federal Ministry for Education, Science and Culture of the Austrian Republic.

Clonal myoblast cultures from skeletal muscle

Rat skeletal myoblasts were isolated from male F344 rats (6 weeks of age). Rats were anaesthetized (ketamine, 0.1 mg (100 g bw)⁻¹, and xylazine, 0.02 mg (100 g bw)⁻¹, i.p.) and hindlimb muscle biopsies (approximately 0.5–1.0 g) removed. The biopsies were minced into pieces of approximately 1 mm³ and enzymatically dissociated as previously described (Blau & Webster, 1981).

Enzymatically isolated single rat skeletal myoblasts were manually collected with a glass micropipette (tip diameter ∼30 μm) under microscopic control and transferred to gelatine-coated wells (96-well plate) containing proliferation medium (minimal essential medium (MEM) supplemented with 15% fetal calf serum (FCS) and 0.1 μg ml⁻¹ gentamicin) as previously described (Baroffio et al. 1996). After the myoblasts had reached confluency the cultures were replated at a lower density. Myotube formation was induced by culturing confluent myoblasts in differentiation medium containing 5% FCS.

Labelling of the clonal myoblast

Rat skeletal myoblasts were transfected with the green fluorescence protein (α-actin–GFP) reporter plasmid containing the neomycin resistance gene. Cationic liposome transfection (FuGen transfection with pECFP–actin, Clontech) resulted in stable GFP expression. GFP-positive (GFP⁺) cells were separated by fluorescence-activated cell sorting and subsequent manual collection of single GFP⁺ cells with a glass micropipette under fluorescence-microscopic control (Baroffio et al. 1996). In some experiments the cells were additionally labelled by the membrane dye PKH using the fluorescence cell linker kit PKH26-GL (Sigma).

Immunocytochemial characterization of the myoblast cultures

Desmin antibodies were used to prove the purity of the clonal myoblast cultures. The cell cultures were fixed with methanol at −20°C for 10 min, followed by two rinses in phosphate-buffered saline (PBS). The staining was performed using Desmin Immunohistology Kit (IMMH-5, Sigma) and monoclonal anti-skeletal myosin (fast) antibodies (clone My-32, Sigma) following the manufacturer's instructions.

Myocardial infarction and cell transplantation

To enable access of the collagenase solution to the infarcted region (see below) we employed a reperfusion infarct model rather than ligating the LAD permanently. Twenty male F344 Fischer rats were anaesthetized (ketamine, 0.1 mg (100 g bw)⁻¹, and xylazine, 0.02 mg (100 g bw)⁻¹, i.p.), placed in a supine position and tracheally ventilated. The heart was exposed through a 2 cm left lateral thoracotomy and the left anterior descending (LAD) coronary artery was ligated with a Prolene 7/0 suture under the distal portion of the left atrial appendix. The surgical knot was laid on a silicon tube to allow firm closure and reopening without significant injury of the target vessel and the overlying cardiac tissue. After 30 min of ischaemia, 200 μl of cell suspension containing about 10⁷ cells were injected into 10 sites in the centre and border zone of the ischaemic area. After haemostasis was achieved the ligature was reopened and the target area was reperfused. The muscle layer and skin incision was closed with Vicryl 3/0 sutures after drainage of the left thoracic cavity with a silicon tube. The animals were monitored for 4 h post-operatively.

Twenty-four hours after transplantation, the PKH⁺–GFP⁺ myoblasts were located in interseptal bulks. Two weeks after, the free wall of left ventricle was substantially thinned representing a fibrous scar. Transplanted PKH⁺–GFP⁺ myoblasts typically aligned with the circumference of the left ventricle (data not shown).

Enzymatic isolation of myoblasts from the myocardium

In order to study their voltage-gated ion channels the injected myoblasts were enzymatically isolated from the myocardium by means of a Langendorff perfusion technique as previously described (Hering et al. 1983). In short, the hearts were removed from anaesthetized rats, retrogradely perfused with a calcium free solution and then perfused for 10–20 min with a collagenase-containing solution. After digestion the infarcted region of the myocardium was dissected and transferred to a Petri dish. Single cells were isolated at different times after implantation (24 and 48 h, 1 and 2 weeks) for subsequent patch clamp studies.

Primary culture of myoblasts isolated from the myocardium

The Langendorff perfusion described above was performed under sterile conditions. The cell suspension was transferred to a culture dish containing proliferating medium (MEM supplemented with 15% FCS and 0.1 μg ml⁻¹ gentamicin) and the cells were held in primary culture for up to 1 week.

Clonal cultures of GFP⁺ myoblast isolated from myocardium

Due to overgrowth by other cell types the labelled myoblasts were difficult to identify after periods longer than 1 week. To overcome this problem we established clonal cultures deduced from single GFP⁺ myoblasts. GFP⁺cells were manually collected from a suspension of detached primary cultured cells as described above. These homogeneous GFP⁺ myoblast cultures were suitable for patch clamp studies. Desmine labelling confirmed the muscle origin of the established clonal cultures (Fig. 6C). Furthermore, myotubes stained positive for fast skeletal myosin heavy chain (MHC) (Fig. 8).

Figure 6. Recovery of sodium and barium currents in cell culture.

A, photomicrograph of a 48 h primary culture of cells isolated from the myocardium 2 weeks after implantation. GFP⁺ myoblasts (arrows) are surrounded by non-fluorescent cells. B, fluorescent image of A. Scale bars in A and B correspond to 25 μm. C, fluorescent image of clonal GFP⁺ rat myoblast culture deduced from a single GFP⁺ cell isolated from the myocardium 2 weeks after implantation. All cells expressed the marker gene. D, the GFP⁺ cultures were stained with antibodies against desmin as described in Methods. All cells were found to be desmin positive. The scale bars in C and D correspond to 30 μm. E and F, the fraction of myoblasts with I_Na and I_Ba steadily increased with time. Insets: representative families of I_Na and I_Ba in GFP⁺ myoblast after 6 weeks in culture. Currents were evoked by the protocols described in Fig. 2. Voltage dependence and current kinetics were indistinguishable from control. The I_Ba inactivation time constant at −20 mV was 12.7 ± 0.5 ms (n = 9) versus 13.4 ± 0.4 ms in control (n = 7, P > 0.05). I_Na inactivated at −20 mV with a time constant of 1.72 ± 0.07 ms (n = 9) versus 1.55 ± 0.07 ms in control (n = 15, P > 0.05).

Figure 8. Skeletal muscle phenotype of myoblasts isolated from the myocardium.

In differentiation medium the GFP-positive cells fused into multinucleated myotubes. The figure illustrates the initial stage of myotube formation (day 1 in differentiation medium). The multinucleated myotube stained positive for MHC (A) and GFP (B). Scale bars correspond to 30 μm.

Patch clamp studies

Voltage-gated ion channels were studied in freshly isolated and cultured myoblasts at 22–25°C using the patch-clamp technique (Hamil et al. 1981) by means of an Axopatch 200A patch clamp amplifier (Axon Instruments, Union City, CA, USA). Patch pipettes with resistances of 1–4 MΩ were made from borosilicate glass (Clark Electromedical Instruments, UK).

Barium currents (I_Ba) through voltage-gated Ca²⁺ channels were measured in an extracellular solution containing (mm): BaCl₂ 10, tetraethylammonium chloride 145, Hepes 10 (buffered to pH 7.4 with methanesulphonic acid). Voltage-gated sodium (I_Na) and potassium (I_K) currents were measured in extracellular solution containing (mm): NaCl 130, MgCl₂ 2, CsCl 5.4, glucose 10, Hepes 10 (buffered to pH 7.4 with NaOH). For I_Ba and I_Na measurements patch pipettes were filled with a solution containing (mm): CsCl 145, MgCl₂ 1, Hepes 10, EGTA0.1 (adjusted to pH 7.25 with CsOH). I_K were measured using intracellular solution containing (mm): KCl 145, MgCl₂ 1, Hepes 10, EGTA 0.1 (buffered to pH 7.2 with NaOH).

Data were digitized using a Digidata 1200 interface (Axon Instruments), filtered (four-pole Bessel filter) at 2 kHz (I_Ba, I_k) and 10 kHz (I_Na) and stored on a computer hard disc.

Statistical analysis was performed using unpaired Student's t test. Data are given as means ± s.e.m. Leak currents were subtracted digitally using average values of scaled leakage currents elicited by a 10 mV hyperpolarizing pulse.

Results

Voltage-gated ion channels in control myoblasts

Before the myoblasts were transplanted into the myocardium we analysed possible effects of GFP transfection on ion channel gating and expression. A typical culture that was established from a single GFP⁺ myoblast is illustrated in Fig. 1A and B. Myoblasts and myotubes were all found to be GFP⁺ (Fig. 1B and C). The ability of GFP⁺ myoblasts to form myotubes is illustrated in Fig. 1C.

Figure 1. Clonal cultures of GFP⁺ myoblasts.

A, phase-contrast image of clonal GFP-transfected rat myoblast culture. B, fluorescence image of the culture shown in A. C, myotube formation in GFP⁺ myoblasts after 5 days in differentiating medium (see Methods). The scale bars correspond to 100 μm.

In control (i.e. non-transfected) and GFP⁺ myoblasts three types of currents were observed: sodium currents (I_Na), transient T-type currents (I_Ba) and potassium currents (I_K). A comparison of the sodium and calcium channel expression, current densities, half-maximum of channel activation and the time constants of channel inactivation at −10 mV in GFP⁺ and control myoblasts is given in Table 1. Table 2 describes key parameters of two types of voltage-gated potassium currents that were observed, transient outward (I_Kto) and ultra-rapid delayed rectifier (I_Kur).

Table 1.

Features of voltage-gated I_Ba and I_Na in GFP⁺ and GFP⁻ myoblasts

	Cells with current (%) (n)	Current density (pA pF−1) (n)	V0.5, act. __(mV) (n)	τinact. at 10 mV (ms) (n)
Ba2+ currents
Control	69 (13)	3.7 ± 1.1 (9)	−30.9 ± 1.1 (9)	13.6 ± 0.5 (9)
GFP+	68 (25)	3.9 ± 1.3 (17)	−31.3 ± 0.9 (15)	13.4 ± 0.4 (15)
Primary	59 (17)	4.1 ± 1.3 (10)	−31.0 ± 1.1 (10)	13.3 ± 0.5 (10)
Na2+ currents
Control	66 (12)	79 ± 9 (10)	−34.2 ± 1.0 (10)	1.61 ± 0.07 (10)
GFP+	64 (22)	82 ± 12 (14)	−34.6 ± 0.9 (14)	1.55 ± 0.07 (14)
Primary	64 (14)	85 ± 11 (9)	−34.9 ± 1.1 (9)	1.56 ± 0.09 (9)

Table 2.

Comparison of the voltage-gated I_K in control and GFP⁺ myoblasts

	Cells with current (%) (n)	Current density at 50 mV (pA pF−1) (n)
IKto currents
Control	77 (22)	86 ± 16 (17)
GFP+	79 (24)	83 ± 14 (21)
Primary	60 (15)	79 ± 17 (9)
IKur currents
Control	9 (22)	81 ± 9 (2)
GFP+	8 (24)	78 ± 14 (21)
Primary	13 (15)	85 ± 10 (2)

Transient (T-type) I_Ba were detected in 69% of control and 68% of GFP⁺ cells (Table 1). A typical family of I_Ba through Ca²⁺ channels recorded in a GFP⁺ myoblast is illustrated in Fig. 2A (left panel). T-type Ca²⁺ channels activated at similar potentials and displayed similar kinetics in GFP⁺ and control cells (Table 1).

Figure 2. Voltage-dependent barium, sodium and potassium currents in cultured myoblasts.

A, I_Ba through T-type Ca²⁺ channels in GFP⁺ myoblasts (left panel). Currents were evoked by 150 ms depolarizing pulses from a holding potential of −100 mV. Corresponding current–voltage relationship of the I_Ba peak is shown in the right panel. B, representative family of I_Na (left panel). Currents were evoked by 10 ms depolarizations from a holding potential of −120 mV. Right panel shows corresponding current–voltage relationship of peak I_Na. C, representative traces of I_Kto (left panel). Currents were evoked by 300 ms depolarizations from a holding potential of −60 mV to indicated test potentials. Right panel: corresponding current–voltage relationship of the peak current. D, representative traces of I_Kur (left panel, same pulse protocol as in C) and corresponding current–voltage relationship of the peak current (right panel).

I_Na were detected in 64% of GFP⁺ and 66% of non-transfected cells (Table 1). A representative I_Na family of a GFP⁺ myoblast is depicted in Fig. 2B (left panel). The voltage of half-maximal current activation, current inactivation kinetics and current densities were similar in GFP⁺ and control myoblasts (Table 1).

In 79% of GFP⁺ cells and in 77% of non-transfected cells we observed transient outward potassium currents (I_Kto). Figure 2C (left panel) shows rapidly inactivating currents of a GFP⁺ cell. The voltage dependence of I_Kto is illustrated in Fig. 2C right panel and the mean I_Kto densities are compared in Table 2. Rapidly activating and slow inactivating voltage-gated potassium currents (I_Kur) were measured in 9% of control and 8% of GFP⁺ myoblasts (Fig. 2D, Table 2).

Our data suggest that permanent GFP transfection did not significantly affect expression of voltage-gated channels or their kinetic properties. In experiments where the cells were isolated shortly after injection (24 and 48 h), myoblasts were labelled with the lipid dye PKH (see methods, Fig. 3A and B). PKH labelling had no effect on ionic currents (data not shown).

Figure 3. Down-regulation of barium currents in transplanted myoblasts.

A, cell suspension after enzymatic isolation (see Methods). The phase-contrast image shows two cardiomyocytes and one myoblast (indicated with arrow) isolated 24 h after implantation. The scale bar corresponds to 50 μm. B, the corresponding fluorescence image highlights the PKH⁺ myoblast. C, percentage of cells positive for Ca²⁺ channel currents in control and complete absence of currents at ≥ 24 h after myoblast injection. D, typical traces during stepwise depolarization of the membrane (same voltage protocol as in Fig. 2B) of a GFP⁺ cell isolated 24 h after implantation.

Prolonged in vitro culturing may change the cell phenotype. We have therefore compared the properties of voltage-gated ion channels in primary and clonal GFP⁺ cultures. Desmin staining revealed that our primary cultures (enriched by filtering through a 10 μm Millipore filter and subsequent pre-plating) consisted of about 80% of myoblasts (not shown). Voltage-gated T-type Ca²⁺ channels were detected in 59% of cells (n = 17), while 64% of cells (n = 14) displayed Na⁺ currents (Table 1). Sodium and calcium channel current densities, half-maximum of channel activation and the time constants of channel inactivation at −10 mV in primary cultures were not significantly different from that of GFP⁺ and control myoblasts (Table 1).

I_Kto and I_Kur (Snyders, 1999) currents were detected in 60% and 13% of cells (n = 15), respectively. K_TO and K_UR current densities and voltage dependencies in primary cultures were not significantly different from that of primary clonal cultures (Table 2). These data suggest similar functional properties of myoblasts in clonal and primary cultures.

Voltage-gated ion channels in myoblasts freshly isolated from the myocardium

The labelled myoblasts were transplanted into the infarcted myocardium as described in Methods. Subsequently the cells were enzymatically isolated by Langendorf perfusion 24 and 48 h, and 1 and 2 weeks after transplantation. The infarcted region was excised from the digested ventricle and transferred to a Petri dish. Small round myoblasts were identified using fluorescence microscopy. Figure 3A shows a representative phase-contrast image of a cell suspension isolated 24 h after cell transplantation.

Twenty-four hours after injection into the infarcted myocardium no barium currents were detectable (n = 29). As illustrated in Fig. 3C and D, the currents did not recover within 2 weeks (n = 29).

I_Na were still detectable in 44% of the cells at 24 h (n = 18) and in 14% at 48 h (n = 28) after implantation (Fig. 4A). One week (n = 19) and 2 weeks n = 26 after transplantation I_Na were completely suppressed (Fig. 4A and B). As shown in Fig. 4C and D, I_Na densities, kinetics and current–voltage relationships in the myoblasts isolated from the myocardium were not significantly different from control (P > 0.05).

Figure 4. Down-regulation of sodium currents in transplanted myoblasts.

A, bar graph illustrating the progressing reduction of the cell fraction with detectable I_Na. Inset: superimposed I_Na (pulse from −120 to −20 mV) of a control myoblast and a myoblast isolated 48 h after implantation. B, representative whole cell recordings from a myoblast isolated 1 week after transplantation (same protocol as in Fig. 2B). C, mean I_Na densities (pA pF⁻¹) at different times after implantation. Current densities estimated 24 and 48 h after injection did not significantly differ from control (P > 0.05, n = 8 and n = 4 for 24 and 48 h, respectively). D, superimposed current–voltage relationships of peak I_Na in control (○), and GFP⁺ myoblasts isolated after 48 h (•) and 1 week (▪) after cell implantation.

I_K of myoblasts isolated from the myocardium displayed exclusively a slow time course of inactivation (14–25% during 300 ms, Fig. 5B) compared to I_Kto in control cells inactivating almost completely.

Figure 5. Down-regulation of potassium currents.

A, I_Kto was detected in a majority of control cells, while only a few cells showed I_Kur. In cells isolated from the myocardium we observed exclusively I_Kur currents and not the fast inactivating currents found in control (Fig. 2C). The percentage of cells with I_Kur gradually declined within 2 weeks. B, typical whole cell recordings from a cell isolated 2 weeks after implantation. I_Kur were evoked by 300 ms test pulses from −60 mV. Traces corresponding to the test potentials of −20, 0, 20 … 80 mV are shown. C, I_Kur densities in cells isolated at different time intervals after cell transplantation compared to control. Current amplitudes were measured during membrane depolarizations from a holding potential of −60 mV to +50 mV. The current densities were not significantly different (P > 0.05) from control (n = 21), 24 h (n = 15), 48 h (n = 12), 1 and 2 weeks (n = 6). D, current–voltage relationship of peak I_Kur of the experiment shown in B.

The percentage of myoblasts positive for voltage-gated I_Kur gradually declined from 83% at 24 h (n = 18) to 29%(n = 21) at 2 weeks after cell injection (Fig. 5A). I_Kur densities in cells isolated 24 and 48 h, and 1 and 2 weeks after injection were, however, not significantly different from control (P > 0.05, Fig. 5C). The voltage dependence of current activation was similar to I_Kto observed in control myoblasts (Fig. 5D, compare to Fig. 2D).

The cell isolation procedure did not affect ionic currents in myoblasts. Incubating control myoblast cultures for 6–20 min in collagenase (0.8 mg ml⁻¹ Worthington Type 2) neither affected current density nor significantly reduced the percentage of cells with I_Na, I_Ba or I_K (data not shown).

Reappearance of ion channels in primary culture

Enzymatically isolated cell suspensions were transferred to culture medium and held for up to 1 week in primary culture. During this period GFP⁺ myoblasts were clearly detectable. Figure 6A illustrates a 24 h primary culture consisting of GFP⁺ myoblasts as well as a large number of non-labelled cells. Myoblast identification at later stages (> 1 week) was complicated due to overgrowth of non-labelled cells. To overcome this difficulty we established clonal cultures from single GFP⁺ cells isolated from the myocardium (see Methods). GFP fluorescence, myotube formation, and desmin and fast skeletal muscle myosin heavy chain (MHC) expression confirmed that these clonal cultures consisted exclusively of myoblasts (Figs 6C and D, 7D and 8).

Figure 7. Recovery of potassium currents in cell culture.

A, time-dependent increase in the number of myoblasts displaying I_K. Only slow inactivating I_Kur currents were detected in cells isolated from the myocardium. Notably, after 6 weeks in culture the fraction of cells with detectable I_K (i.e. I_Kur) was not statistically different from control cells expressing either type of I_K (P > 0.05). B, representative family of I_Kur in a myoblast culture 6 weeks after cell isolation. Currents were evoked by 300 ms depolarizations from −60 mV to 90 mV with 10 mV increments. C, peak current–voltage relationship of the experiment shown in B. D, myotube formation in GFP⁺ myoblasts in a clonal culture isolated from myocardium (see Methods). The picture represents superposition of phase-contrast and fluorescence images. Blue colour highlights Dapi stained nuclei. The scale bar corresponds to 50 μm.

The percentage of cells expressing calcium channels gradually increased with time. Twenty-four hours after isolation I_Ba were observed in 19% of GFP⁺ cells. After 6 weeks the fraction of I_Ba-positive cells (70%, n = 17) reached the control level (68%, n = 25). Figure 6E (inset) illustrates a typical family of voltage-gated I_Ba in a 6 week clonal culture of GFP⁺ myoblasts isolated from the myocardium. I_Ba kinetics and current density did not differ from control (data not shown).

Similar observations were made for the recovery of I_Na. After 6 weeks in culture the fraction of cells positive for I_Na had recovered to about 70% (n = 17, Fig. 6F). Current activation, the potential corresponding to the maximum of the current–voltage relationship and the time constant of I_Na inactivation at −20 mV were almost identical to control (Table 1, Fig. 2B). After 6 weeks in cell culture, mean I_Na density (72 ± 12 pA pF⁻¹, n = 12) was not significantly different from control (82 ± 12 pA pF⁻¹, n = 14, P > 0.05, Table 1). However, as illustrated in Fig. 6F, I_Na recovered at a slower time course than I_Ba.

In contrast to the complete down-regulation of I_Ba and I_Na, I_K were detectable 2 weeks after transplantation. These channels recovered faster. After 48 h the percentage of I_K-positive cells had reached the control level (Fig. 7A). After 24 h the current density of I_Kur (80 ± 13 pA pF⁻¹, n = 14) was statistically indistinguishable from control (P > 0.05). Interestingly, even after 6 weeks under culture conditions I_K in myoblasts isolated from the myocardium resembled slow inactivating I_Kur detected in only a minority of control cells (Figs 2C and D).

Discussion

In the present study we have analysed for the first time the functional properties of skeletal muscle myoblasts that had been transplanted into the infarcted myocardium. We report here, that the infarcted area contains a significant number of mononucleated myoblasts for up to 2 weeks after implantation. These cells were enzymatically isolated by means of a standard Langendorff perfusion protocol. The yield of GFP⁺ or PKH⁺ cells enabled a detailed characterization of voltage-gated ion channels at different time intervals after implantation (Figs 3–7).

The skeletal muscle origin of the isolated cells is indicated by: (i) GFP staining (permanently GFP-transfected myoblasts were injected, Figs 3A, 6B and D, and 8B), (ii) fusion competence (a hallmark of skeletal myoblasts, Figs 7D and 8), and (iii) expression of fast skeletal myosin heavy chain in myotubes (Fig. 8).

Transdifferentiation of myoblasts into cardiomyocytes does not occur (Reinecke et al. 2002). In the myocardium myoblasts form multinucleated cross-striated muscle fibres expressing MHC but not the cardiac-specific markers α-MHC, cardiac troponin I or the atrial natriuretic peptide (Reinecke et al. 2002). The GFP⁺ cells that were analysed are unlikely to represent skeletal–cardiac hybrid cells that have recently been described (Reinecke et al. 2004). Hybrid cells would be expected to express T- and L-type channels. We observed, however, only T-type channels (Figs 2A and 6E). Furthermore, fused cells would appear as cardiac myocytes (Reinecke et al. 2004) rather than round GFP⁺ myoblasts (Fig. 3A). In vivo fusion is a rare event and it is therefore highly unlikely that the presence of hybrid cells substantially affects the interpretation of our data.

Interestingly, in none of the 24 cell isolations did we detect multinucleated myotubes (or myofibres) suggesting that a refinement of the enzymatic treatment will be required for their isolation and characterization.

Permanent GFP transfection was particularly suitable for cell identification in primary cultures after up to 6 weeks (i.e. after multiple cell divisions), whereas PKH fluorescence enabled myoblast recognition for up to 48 h after cell transplantation. GFP transfection or PKH labelling affected neither the voltage-gated ion channels nor the ability of myoblasts to form myotubes (Tables 1 and 2).

Down-regulation of voltage-gated ion channels in transplanted myoblasts

The majority of control myoblasts expressed voltage-gated sodium, T-type calcium and transient outward potassium channels (Tables 1 and 2). In a small population we observed, however, ultra-rapid delayed rectifier channels (Fig. 2D). The properties of these four channel types were found to be similar to channels previously described in myoblasts of other species (Hamann et al. 1994; Liu et al. 1999; Snyders, 1999).

In the present study we found a progressive down-regulation of inward and outward currents in myoblasts implanted into the myocardium. While I_Ba disappeared within 24 h I_K were present up to 2 weeks after transplantation. The individual time courses of down-regulation suggest that the underlying mechanism might be different for each channel type (Figs 3–5). In none of the experiments did we detect slowly activating calcium channel currents that are typical for skeletal muscle myotubes (Beam & Knudson, 1988) confirming that patch clamp studies were carried out exclusively on mononucleated myoblasts.

Bernheim and coworkers have clearly demonstrated that T-type Ca²⁺ channels play an essential role in myoblast fusion (Liu et al. 1999; Bijlenga et al. 2000). It is therefore tempting to speculate that down-regulation of this channel type in the infarcted myocardium prevents myoblast fusion at later stages after cell implantation.

Recovery of ion channels under cell culture conditions

Two weeks after implantation we observed neither sodium nor calcium channels in GFP⁺ myoblasts (Figs 3 and 4). The population of channels expressing potassium channels decreased about threefold (Fig. 5).

On the one hand down-regulation of ion channels were caused by acute cellular stress underlying the ischaemia and reperfusion associated injury of the infarcted myocardium such as hypoxia, acidification, release of inflammatory cytokines, activation of the complement system, disturbances of Ca²⁺ homeostasis and the release of reactive oxygen species (Kukreja & Janin, 1997; Berridge et al. 1998; Pinto & Boyden, 1999; Ermak & Davies, 2002). In such a scenario one would expect, however, faster recovery (e.g. within several hours) under cell culture conditions.

On the other hand the down-regulation might reflect substantial changes in gene expression. This hypothesis is indirectly supported by our finding that in cells isolated from the myocardium we observed exclusively I_Kur currents and not the fast inactivating current observed in control (Fig. 2C).

The apparent switch in potassium channel expression could alternatively reflect a selection mechanism within the infarcted myocardium favouring a subpopulation of myoblasts expressing ultra-rapid delayed rectifier channels (Fig. 2C and D).

Down-regulation of voltage-gated ion conductances in the infarcted region was previously described for myocardial cells. These studies suggest that transcription of different ion channel proteins in the myocardium is affected by pathological conditions such as a myocardial infarction (see Pinto & Boyden, 1999 for review). It cannot be excluded that myoblasts implanted into the infarcted myocardium undergo similar changes.

We were interested in whether the down-regulation of ion channels was reversible. Stable GFP expression enabled us to establish the time course of reappearance of ion currents. As shown in Figs 6 and 7 recovery of the different channel types occurred at different time courses (between 2 days and 6 weeks). The slow time courses of down-regulation of calcium, sodium and potassium channels and their slow recovery would argue for alterations in gene expression rather than an acute modulation of channel function by metabolic stress.

Potential role of skeletal myoblasts in the myocardium

Grafting of autologous skeletal muscle myoblasts improves regional myocardial performance in vivo (Taylor et al. 1998; Jain et al. 2001). These and other studies suggest that ventricular remodelling after a myocardial infarction can be reduced by implantation of skeletal myoblasts (Taylor et al. 1998; Reinecke & Murry, 2003; Van Den Bos & Taylor, 2003; Menasché, 2003).

Contraction studies on myocardial wound strips and immunohistochemical analysis demonstrate, however, that the newly formed fibres keep essential characteristics of skeletal muscle such as tetanic contraction and expression of the skeletal muscle myosin heavy chain. The grafts do not express cardiac-specific markers such as the α-myosin heavy chain, cardiac troponin I or the atrial natriuretic factor (see Murry et al. 2002 for review).

More recent studies reveal that newly formed myofibres are electrically isolated from surrounding myocardial cells and contract independently from neighbouring cardiomyocytes (Murry et al. 2002; Leobon et al. 2003). It is difficult to imagine that muscle cells that are not electrically coupled will contribute synchronous contractility. Alternatively engrafted myoblasts may affect the cardiac function through paracrine effects mediated by release of growth factors such as hepatocyte growth factor (HGF) (Menasché, 2003).

In summary, we have analysed for the first time the biophysical properties of myoblasts after engraftment into the infarcted myocardium. Sodium, potassium and calcium channels were found to be down-regulated compared to myoblasts before transplantation. Our data suggest that these cells enter a ‘hibernating state’ of low excitability. Down-regulation of T-type channels, a key channel type for cellular fusion (Liu et al. 1999; Bijlenga et al. 2000), would imply that myoblasts lose fusion competence during engraftment.

Our study demonstrates that these myoblasts are capable of re-entering the cell cycle. It is therefore tempting to speculate that in analogy to skeletal muscle, some of the injected myoblasts become quiescent cells retaining their proliferative potential.

The signals of the myocardial environment affecting the ion channel expression in skeletal myoblasts are presently unknown. In the C2 cell line sodium and calcium channels were reversibly down-regulated in vitro by addition of a single mitogen (transforming growth factor β-1) to the culture medium (Caffrey et al. 1989). Further analysis of functional properties of myoblasts isolated at different time points after transplantation will unravel the factors responsible for the observed functional changes and pave the way for deeper understanding of the molecular basis of myoblast differentiation or dedifferentiation in a myocardial environment. Future studies will clarify which factors (e.g. proinflammatory cytokines in the infarcted environment, hypoxias and related changes in cell metabolism or changes in gene expression) induce the observed down-regulation of ion channels and if these factors have individual effects on different channel types. At this stage we cannot, however, exclude that all or some of the observed down-regulation of voltage-gated Na⁺, K⁺ and Ca²⁺ currents may take place in healthy myocardium as well.