Beta2-adrenergic receptor overexpression in the developing mouse heart: evidence for targeted modulation of ion channels

Abstract

1. We studied the effect of overexpression of the beta2-adrenergic receptor (beta2-AR) in the heart on ion channel currents in single cells isolated from hearts of fetal and neonatal transgenic and wild-type mice. The beta2-AR transgene construct was under the control of the murine alpha-myosin heavy chain (alpha-MHC) promoter, and ion channel activity was measured at distinct developmental stages using whole-cell and perforated patch clamp techniques. 2. We found no change in L-type Ca2+ channel current (ICa) density in early embryonic stages (E11-13) of beta2-AR transgenic positive (TG+) mice, but significant increases in ICa density in intermediate (E14-16, 152 %) and late (E17-19, 173.7 %) fetal and neonatal (1 day post partum, 161 %) TG+ compared with transgenic negative (TG-) mice. This increase in ICa was accompanied by a negative shift in the peak of the current-voltage relationship in TG+ mice. 3. Transient (< 3 min) or prolonged (16-24 h) exposure of TG- neonatal stage myocytes to 8-Br-cAMP (300 microM) increased ICa density and caused a shift in the current-voltage relationship to a similar extent to that seen in TG+ mice. In TG+ myocytes, 8-Br-cAMP had no effect. Exposure of TG+ cells to Rp-cAMPS reversed both the shift in voltage dependence and reduced the peak current density observed in these myocytes. We concluded from these results that the L-type Ca2+ channel is maximally modulated by cAMP-dependent protein kinase (PKA) in TG+ mice and that the alpha-MHC promoter is functional in the ventricle as early as embryonic day 14. 4. In contrast, we found that slow delayed rectifier K+ channels were not changed significantly at any of the developmental stages studied by the overexpression of beta2-ARs compared with TG- mice. The sensitivity of murine slow delayed rectifier K+ channels to cAMP was tested by both transient and prolonged exposure to 8-Br-cAMP (300 microM), which increased the slow delayed rectifier K+ channel current (IK,s) density to a similar extent in both TG- and TG+ neonatal myocytes. In addition, we found that there was no difference in the concentration dependence of the response of ICa and IK,s to 8-Br-cAMP. 5. Thus, overexpression of the beta2-AR in the heart results in distinct modulation of ICa, but not IK,s, and this is not due to differences in the 8-Br-cAMP sensitivity of the two channels. Instead, these results are consistent with both compartmentalization of beta2-AR-controlled cAMP and distinct localization of L-type Ca2+ and slow delayed rectifier K+ channels. This cAMP is targeted preferentially to the L-type Ca2+ channel and is not accessible to the slow delayed rectifier K+ channel.

Figure 1. β₂-AR overexpression increases I_Ca in the developing mouse heart

A, current traces illustrating families of currents (for voltages, see Methods) measured in cells from TG- and TG+ early stage embryonic (left) and neonatal (right) hearts. B, bar graph summarizing peak I_Ca density recorded at +10 mV from a large number of cells (indicated above each bar) for three embryonic stages and for neonatal cells. * Significant difference compared with TG-, P < 0.01.

Figure 2. Voltage-dependent changes in I_Ca in TG+ cells

A, mean peak current-voltage relationships for TG+ ( n = 25) and TG- ( n = 20) cells. Note that the peak of the relationship occurred near +5 mV for TG+ and near +15 mV for TG- cells. The current traces (top) illustrate typical TG- (left) and TG+ (right) recordings (see Methods for voltage protocol). B, conductance- voltage relationships determined from A by normalizing currents to driving force. The smooth curves are Boltzmann fits to the data with the following parameters: potential at half-maximal activation (V_½) = -7 mV, slope factor (V_K) = 5.2 mV (TG+); V_½ = 1.8 mV, V_K = 6.5 mV (TG-). C, β₂-AR overexpression did not affect I_Ca steady-state inactivation. Steady-state inactivation was measured using 5 s conditioning pulses for TG+ and TG- cells. The smooth curves are Boltzmann relationships: potential at half-maximal inactivation (V_½) = -8.6 mV; V_K = 5.8 mV.

Figure 3. Influence of 8-Br-cAMP on I_Ca in TG- and TG+ cells

A, transient exposure to 8-Br-cAMP (300 μM) reversibly increased the peak I_Ca (activated by repeated 40 ms depolarizations to +10 mV) in a TG- cell. Typical Ca²⁺ channel currents shown above the plot were sampled at the points indicated by the arrows. B, mean current-voltage relationships for I_Ca in TG- cells before (Control) and after overnight (16-24 h) exposure to 8-Br-cAMP (n = 4-6). C, mean current-voltage relationships for I_Ca in TG+ cells in which overnight exposure to 8-Br-cAMP had no significant effect (P > 0.05; n = 6-18).

Figure 4. Comparison of the effects of β₂-AR overexpression and 8-Br-cAMP (300 μM) on peak I_Ca

Bar graphs summarizing peak current densities recorded at +10 mV. Left, peak currents in TG- (n = 26) vs. TG+ (n = 30) cells. Middle, peak current density in TG- cells before and after transient (< 3 min; middle left, n = 6 each) and prolonged (16-24 h; middle right, n = 8 each) exposure to 8-Br-cAMP. * There was no significant difference between the enhancement of peak I_Ca in the TG+ cells (left) and that produced by 8-Br-cAMP in TG- cells (middle; unpaired t test, P > 0.05). Right, peak current density before and after prolonged exposure of TG+ cells to the same concentration of 8-Br-cAMP.

Figure 5. Effect of Rp-cAMPS on I_Ca recorded from TG+ cells

Mean ( n = 6) peak current-voltage relationships for currents measured in TG+ cells before (Control) and after exposure to Rp-cAMPS (300 μM). Steady-state effects which were obtained within 3-5 min of exposure to the PKA inhibitor are shown. Note that the peak of the current-voltage relationship was shifted to positive voltages and reduced in amplitude.

Figure 6. β₂-AR overexpression did not affect I_K,s

Right, representative traces of I_K,s (activated by isochronal 2 s depolarizations from -40 to +60 mV in 20 mV increments from a holding potential of -40 mV) recorded from TG- (top) and TG+ (bottom) cells. Left, the mean current-voltage relationships for time-dependent I_K,s activated during depolarization for TG- ( n = 19) and TG+ ( n = 20) cells (neonatal stage). There was no significant difference between TG- and TG+ currents at any voltage.

Figure 7. Effects of 8-Br-cAMP on I_K,s in TG+ and TG- cells

A shows mean I_K,s density measured at +40 mV in TG- and TG+ cells in the absence of 8-Br-cAMP (Control) and after 16-24 h incubation in the presence of 8-Br-cAMP (300 μM), n = 5-18. Representative I_K,s traces (at +60 mV) before and after exposure to cAMP in TG- and TG+ cells are shown above the bars. * Significant difference compared with control, P < 0.01. B shows the relative I_K,s amplitude before (Control) and after transient (< 3 min) exposure to 300 μM 8-Br-cAMP in TG- and TG+ cells. Currents were normalized to those in control (n = 2-6). * Significant difference compared with control, P < 0.01.

Figure 8. Concentration dependence of I_K,s and I_Ca response in TG- cells to 8-Br-cAMP

A, bar graph summarizing the normalized change in I_Ca (measured at +10 mV) following transient and 16-24 h (overnight) exposures to 8-Br-cAMP and the change in I_K,s following 16-24 h exposure to 8-Br-cAMP (measured at +40 mV) as a function of [8-Br-cAMP]. There was no significant difference between groups at each 8-Br-cAMP concentration (P > 0.3). B, log concentration-response curves for I_Ca and I_K,s (normalized change in peak I_Ca for transient and 16-24 h exposures and in peak I_K,s for 16-24 h exposure plotted against [8-Br-cAMP]).