Phorbol ester and endothelin-1 alter functional expression of Na+/Ca2+ exchange, K+, and Ca2+ currents in cultured neonatal rat myocytes

Abstract

Endothelin-1 (ET-1) and activation of protein kinase C (PKC) have been implicated in alterations of myocyte function in cardiac hypertrophy and heart failure. Changes in cellular Ca2+ handling and electrophysiological properties also occur in these states and may contribute to mechanical dysfunction and arrhythmias. While ET-1 or PKC stimulation induces cellular hypertrophy in cultured neonatal rat ventricular myocytes (NRVMs), a system widely used in studies of hypertrophic signaling, there is little data about electrophysiological changes. Here we studied the effects of ET-1 (100 nM) or the PKC activator phorbol 12-myristate 13-acetate (PMA, 1 μM) on ionic currents in NRVMs. The acute effects of PMA or ET-1 (≤30 min) were small or insignificant. However, PMA or ET-1 exposure for 48-72 h increased cell capacitance by 100 or 25%, respectively, indicating cellular hypertrophy. ET-1 also slightly increased Ca2+ current density (T and L type). Na+/Ca2+ exchange current was increased by chronic pretreatment with either PMA or ET-1. In contrast, transient outward and delayed rectifier K+ currents were strongly downregulated by PMA or ET-1 pretreatment. Inward rectifier K+ current tended toward a decrease at larger negative potential, but time-independent outward K+ current was unaltered by either treatment. The enhanced inward and reduced outward currents also result in action potential prolongation after PMA or ET-1 pretreatment. We conclude that chronic PMA or ET-1 exposure in cultured NRVMs causes altered functional expression of cardiac ion currents, which mimic electrophysiological changes seen in whole animal and human hypertrophy and heart failure.

Fig. 1.

Exposure of 1 μM PMA or 0.1 μM endothelin-1 (ET-1) for 48–72 h significantly increased cell membrane capacitance (C_m). PMA augmented C_m from 66 ± 4 to 146 ± 8 pF (*P < 0.05, n = 20). ET-1 increased C_m from 69 ± 5 to 86 ± 5 pF (*P < 0.05, n = 21) (4 preparations). Ctl, control; n, number of cells. *P < 0.05.

Fig. 2.

Acute effects of PMA (A) and ET-1 (B) on L-type Ca²⁺ current (I_Ca,L). Incubation for 30 min of PMA or ET-1 did not affect peak I_Ca,L appreciably (3 preparations).

Fig. 3.

Chronic effects of ET-1 on I_Ca,L and T-type Ca²⁺ current (I_Ca,T). Representative traces from I_Ca,L (A) and I_Ca,T (B) during control and after pretreatment with 0.1 μM ET-1 for 48–72 h. C and D: current-voltage relationships for I_Ca,L and I_Ca,T, respectively, showing a significantly increased peak current at the highest values (3 preparations).

Fig. 4.

Measurement of Na⁺/Ca²⁺ exchange current (I_Na/Ca). A: voltage protocol. Starting at a holding potential of −90 mV follows a step to −45 mV to inactive Na⁺ current and then another step to 0 mV to inactive I_Ca,L. Finally, a ramp from +80 to −140 mV was used to assess the Ni-sensitive I_Na/Ca. B: raw traces obtained during control conditions and after application of 5 mM Ni. C: current record obtained after subtraction of the 2 traces. I_Ca, Ca²⁺ current.

Fig. 5.

Effect of PMA and ET-1 on I_Na/Ca. A: acute effect of 10–30-min exposure to 1 μM PMA. No significant changes on I_Na/Ca can be noticed. B: similarly, no effects can be seen by 10–30-min exposure to 0.1 μM ET-1. C: PMA pretreatment for 48–72 h induces a significant increase on I_Na/Ca. D: likewise, ET-1 pretreatment induces a significant (although smaller) increase on I_Na/Ca. In all cases, pipette [Ca²⁺] was 100 nM (4 preparations). Circles are control, triangles are PMA treatment, and squares are ET-1 treatment (white symbols, acute; and black symbols, chronic). *P < 0.05.

Fig. 6.

Effects of PMA and ET-1 on transient outward K⁺ current (I_to). A: raw traces obtained after applying a voltage protocol in control conditions. Two components can be identified: a time-dependent component right after the stimulus and a steady-state component at the end of the pulse. B: raw traces obtained after pretreatment with PMA. C: transient component is severely reduced with PMA or ET-1 treatment. D: steady-state component, conversely, is unaffected by either PMA or ET-1 (3 preparations). I_SS, time-dependent outward K+ current. *P < 0.05.

Fig. 7.

Chronic effect of PMA and ET-1 on delayed rectifier K⁺ current (I_Ks). A: raw current traces obtained under control conditions. B: effect of pretreating neonatal cells for 48–72 h with 1 μM PMA. C: slowly activating component of I_Ks is significantly depressed by 0.1 μM ET-1. D: likewise, PMA decreased significantly I_Ks. E: tail currents are also reduced by ET-1. F: similar effect on tail currents is obtained by pretreatment with PMA (4 preparations). *P < 0.05.

Fig. 8.

Chronic effects of PMA and ET-1 on inward rectifier K⁺ current (I_K1). A: raw data obtained under control conditions. B: current traces obtained after pretreatment for 48–72 h with 0.1 μM ET-1. C: similar pretreatment with 1 μM PMA produced a slight increase (not statistically significant) on I_K1 at more negative potentials. D: similar tendency on I_K1 is observed with ET-1 (4 preparations).

Fig. 9.

Chronic effects of PMA and ET-1 on action potential duration (APD). A: raw action potential measurements for control, PMA, and ET-1 pretreatment. B: mean APD at 80% repolarization (APD₈₀) values for 5 cells each from 2 preparations. Control APD₈₀ = 252 ± 11.7, PMA APD₈₀ = 342 ± 26.9, and ET-1 APD₈₀ = 279 ± 7.5 ms (*P < 0.05 vs. control). V_m, membrane potential.