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.

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.

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).

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.

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.

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.

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 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.

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.