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.

endothelin-1 (ET-1) and protein kinase C (PKC) activation have been implicated in altered cell signaling and gene expression in cardiac hypertrophy and heart failure (2, 10, 11, 43, 45, 56). Neonatal rat ventricular myocytes (NRVMs) in primary culture have been used extensively as a model system to explore the molecular and cellular events responsible for hormonally induced cellular hypertrophy and contractile protein gene expression. In addition, many of the features of pressure overload-induced myocyte hypertrophy observed in vivo can be simulated using these cultured cells. For instance, the exposure of NRVMs to various neurohumoral agents (e.g., adrenergic agonists, ET-1, or angiotensin II) produces myocyte hypertrophy and changes in gene expression characteristic of hypertrophic myocardium in vivo (22, 31, 44, 59, 62). Several of these growth-promoting stimuli lead to the activation of one or more of the isoenzymes of PKC (19, 20, 33, 43, 52, 70). The direct activation of Ca²⁺-dependent and novel PKC isoenzymes by phorbol 12-myristate 13-acetate (PMA) in NRVMs also mimics aspects of the hypertrophic response to pressure overload in vivo. For example, PMA induces the expression of immediate early genes (15) and secondary response genes such as atrial natriuretic factor (58), β-myosin heavy chain (24, 54), and α-skeletal actin (29). PMA exposure also increases the overall cell protein expression (65) yet decreases the expression level and function of the sarco(endo)plasmic reticulum Ca²⁺-ATPase 2a (SERCA2a) in NRVMs (8, 48, 50). The latter may be related to the downregulation of SERCA2 seen in many models of hypertrophy and heart failure (1, 51), contributing directly to mechanical cardiac dysfunction. Ca²⁺-dependent PKC isoenzymes (PKC-α, -β, and -γ) do not appear necessary for the induction of cardiac hypertrophy in response to pressure overload in vivo, but the PKC-α isoenzyme may negatively regulate sarcoplasmic reticulum Ca²⁺ load and contractility, thereby affecting cardiac performance during the transition to heart failure (32). These data indicate that the PKC regulation of SERCA2 and Ca²⁺ handling is important for hypertrophy and heart failure in vivo, yet the effects of PKC activation on Ca²⁺ currents have not been examined in the NRVM model.

ET-1 expression is increased in animal models of cardiac hypertrophy, working in part through the activation of PKC (27, 45). ET-1 also potently stimulates NRVM hypertrophy (9, 61) and is partly dependent on the activation of a novel PKC-ϵ (21). Chronic ET-1 stimulation produces increased cell size and protein synthesis, increased transcription of myosin light chain-2, and atrial natriuretic factor, as well as enhanced sarcomeric assembly (14, 16, 36). Therefore, ET-1 and PKC activation are likely to be critical modulators of protein expression and phenotype in cardiac hypertrophy and heart failure.

Cardiac hypertrophy and failure are also characterized by alterations in electrophysiological properties, notably decreased transient outward K⁺ current (I_to), reduced inward rectifier K⁺ current (I_K1), modestly increased L-type and T-type Ca²⁺ currents (I_Ca,L and I_Ca,T, respectively), and enhanced Na⁺/Ca²⁺ exchange (NaCaX) (3, 6, 14, 39, 42, 46, 60, 64, 68). Indeed, these electrophysiological alterations may be important in triggered arrhythmias in heart failure. There is little information to date concerning how PKC activation alters ionic currents in NRVMs, although Gaughan et al. (17) found that chronic α-adrenergic activation in NRVMs altered the functional expression of Ca²⁺ current (I_Ca) and certain K⁺ currents. In addition, acute (<1 h) treatment of NRVMs with ET-1 was reported to activate reverse-mode NaCaX secondary to Na⁺/H⁺ exchange activation; however, the direct effects on NaCaX activity were not identified in response to ET-1 (14).

The aim of the present study is to characterize alterations in ionic current expression in NRVMs exposed to ET-1 and PMA. We measured the acute effects, which occur in 10–30 min (possibly because of the direct effects PKC-dependent phosphorylation), as well as the long-term effects induced by 48–72 h of exposure (referred to as “chronic treatment”) and then withdrawal during current measurements (which presumably reflect alterations in the functional expression of channel proteins or modulators). We measured I_Ca,L and I_Ca,T, NaCaX current (I_Na/Ca), I_to, I_K1, and delayed rectifier K⁺ current (I_Ks). The acute effects were minimal, whereas chronic PMA and ET-1 exposure produced cellular hypertrophy and altered current densities in a manner consistent with reports in animal models of hypertrophy and heart failure.

METHODS

NRVM isolation and culture.

All animal protocols were approved by the Animal Care and Use Committee at Loyola University Chicago and University of California Davis. Animals used in these experiments were handled in accordance with National Institutes of Health's Guide for the Care and Use of Laboratory Animals (NIH Publication No 85-23, Revised 2010). NRVMs were isolated from the hearts of 1–3-day-old Sprague-Dawley rat pups via collagenase digestion as previously described (54). Dissociated cells were preplated for 1 h in serum-free PC-1 medium (BioWhittaker, Walkersville, MD) to selectively remove nonmuscle cells. Myocytes were then plated in PC-1 medium at a density of 400 or 1,600 cells/mm² onto collagen-coated plastic 35- or 100-mm dishes or chamber slides and left undisturbed in a 5% CO₂-95% room air incubator for 14–18 h. Unattached cells were removed by aspiration, and the attached cells were maintained in a solution of DMEM-medium 199 (4:1; GIBCO, Grand Island, NY) containing antibiotic/antimycotic solution. Ca²⁺-free and Mg²⁺-free Hanks' balanced salt solution, acid soluble calf skin collagen, and antibiotic/antimycotic solution were obtained from Sigma Chemical (St. Louis, MO). PMA (1 μM ) or ET-1 (100 nM) was added at this time, and for parallel control plates medium was refreshed without added PMA or ET-1. Medium was changed daily, and all electrophysiological studies were 48–72 h after the PMA/ET-1 addition time.

For studies of acute exposure to PMA or ET-1, control cells were used and recordings were made before and after 10–30-min exposure to 1 μM PMA or 100 nM ET-1. For chronic studies, the cells exposed for 48–72 h to PMA or ET-1 (or not for control) were studied after the removal of PMA or ET-1 from the medium for 1 to 2 h. This was done to minimize the potentially complicating direct acute effects of PMA or ET-1, allowing an assessment of altered current as a consequence of altered protein expression.

Electrophysiological measurements.

All currents were recorded in whole cell ruptured patch voltage-clamp mode (Axopatch 200) at room temperature, except I_Na/Ca (measured at 35°C) with pipettes of 1–4-MΩ resistance with partial series resistance compensation (71). Ca²⁺ channel currents (L and T type) were measured with Na⁺ and K⁺ absent. The bathing solution contained (in mM) 140 tetraethylammonium (TEA)-Cl, 6 CsCl₂, 2 MgCl₂, 5 CaCl₂, 10 HEPES, and 10 glucose (pH 7.4), adjusted with TEA-OH at room temperature. The pipette solution contained (in mM) 125 CsCl, 10 Mg²⁺-ATP, 20 HEPES, 10 EGTA, 0.3 GTP, and 10 TEA-Cl (pH 7.4), adjusted with CsOH at room temperature. I_Ca,L was measured from a holding potential (E_h) = −50 mV, with depolarizing steps of 10 up to +60 mV (200-ms duration). I_Ca,T was measured from E_h = −100 mV with 2 μM nifedipine to block I_Ca,L with depolarizing steps of 10 up to +60 mV (200-ms duration).

I_Na/Ca was measured as the current blocked by Ni (5 mM) under conditions where most other currents were blocked. The bath solution contained (in mM) 140 NaCl, 6 CsCl, 1 MgCl₂, 5 HEPES, 10 glucose, 2 CaCl₂, and 0.03 2,3-butanedione monoxime (pH 7.4) at 35°C, and the pipette solution contained (in mM) 14 NaCl, 55 Cs-methanesulfonate, 45 CsCl, 10 ATP-Tris, 0.3 GTP-Tris, 5 BAPTA, 5 Br₂-BAPTA, 2.21 CaCl₂, and 1.08 MgCl₂ (pH 7.3) at 35°C. This solution is expected to have a free [Ca²⁺] = 100 nM, and additional experiments were also performed where free [Ca²⁺] in the pipette solution was increased to 1 μM.

K⁺ currents (I_to, I_Ks, and I_K1) were measured using a bath solution containing (in mM) 138 NaCl, 5 KCl, 2 CaCl₂, 1 MgCl₂, 0.3 CdCl₂, 10 HEPES, and 10 glucose (pH 7.4) with NaOH at room temperature. The pipette solution contained (in mM) 60 KCl, 70 K-aspartate, 10 Mg²⁺-ATP, 10 EGTA, 0.3 GTP, and 10 HEPES (pH 7.2) with KOH at room temperature. To record I_to, E_h was set at −70 mV with a 40-ms step to −60 mV, followed by depolarizing pulses from −30 to +120 mV for 300 ms (in 10-mV increments). The rapidly inactivating component was attributed to I_to (and was completely suppressed by 2 mM 4-aminopyridine), whereas the sustained component we refer to here is time-independent outward K⁺ current (I_SS). To record I_Ks, E_h was −50 mV, followed by 10-mV depolarizing pulses from −20 to +80 mV for 3 s, returning to −25 mV for 4.5 s and finally to E_h. For I_K1, E_h = −50 mV followed by 10 mV steps from −140 mV to 0 mV during 300 ms, returning to E_h afterward. Customized software was written to analyze the K⁺ currents.

Currents were expressed as current density by normalizing to cell capacitance, measured at the start of each experiment as described previously (71). Briefly, the capacitative transient due to 5-mV steps was measured and analyzed as C_m = τ_c/ΔV_m[I_o/1 − (I_∞/I_o)], where C_m is the membrane capacitance, τ_c is the time constant of the capacitative current relaxation, I_o is the peak capacitative current determined by single exponential fit and extrapolation to the first sample point after the voltage step ΔV_m, and I_∞ is the amplitude of the steady-state current.

For action potential (AP) recordings, patch pipettes were backfilled with amphotericin (200 μg/ml). The pipette solution contained (in mM) 120 K⁺-glutamate, 25 KCl, 1 MgCl₂, 1 CaCl₂, and 10 N-2-hydroxyethylpiperazine-HEPES (pH 7.4) (KOH). The external solution contained (in mM) 138 NaCl, 4 KCl, 1 MgCl₂, 2 CaCl₂, 0.33 NaH₂PO₄, 10 glucose, and 10 HEPES (pH 7.4) (NaOH).

Western blot analysis from cultured rat neonatal cardiomyocytes.

Adherent myocytes were collected with trypsin-EDTA and resuspended in radioimmunoprecipitation assay buffer supplemented with a protease inhibitor cocktail (Roche). Myocytes were briefly homogenized with a tissue grinder, and protein concentrations were determined by BCA Protein Assay (ThermoScientific). Protein (30 μg/well) were run on a 4–20% gradient polyacrylamide gel at 80 V. Protein samples were transferred to nitrocellulose membranes overnight at 40 V. Membranes were blocked in 5% milk in TBS-Tween-20 for 1 h and incubated with primary antibodies overnight at 4°C with rocking. Primary antibodies for Na⁺/Ca²⁺ exchanger and GAPDH (Fitzgerald) were used at dilutions of 1:500 and 1:4,000, respectively. Horseradish peroxidase secondary antibodies (GE) were incubated at 1:4,000 for 1 h at room temperature. Membranes were visualized with Super Signal West Pico Chemiluminescent Substrate (ThermoScientific).

Compared values were judged different if unpaired Student's two-sided t-test or one-way ANOVA resulted in P ≤ 0.05. Values are expressed as means ± SE.

RESULTS

PMA and ET-1 treatment produce cellular hypertrophy.

NRVMs have been used to model myocardial hypertrophy in response to neurohumoral agents, and optical methods indicated that ET-1 treatment for 24 h increases cell surface area (21). To further investigate the capacity of PKC activation and ET-1 treatment to induce hypertrophy in NRVMs, the cells were treated with PMA or ET-1 and the cell surface area was assessed by measuring cell C_m. Figure 1 shows that 48–72 h exposure to 1 μM PMA or 0.1 μM ET-1 led to a significant increase in cell capacitance compared with control cells cultured for the same period of time. For PMA, the capacitance increased from 66 ± 4 to 146 ± 8 pF (P < 0.05, n = 20 cells), and for ET-1, C_m increased from 69 ± 5 to 86 ± 5 pF (P < 0.05, n = 21 cells). This indicates that the cell surface area is increased by either treatment and is consistent with cellular hypertrophy caused by PKC activation in response to PMA exposure (59) or the hypertrophic effects of ET-1 (18).

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.

Effects of PMA and ET-1 on I_Ca.

Because Ca²⁺ currents are increased during hypertrophy and heart failure (64, 68), we examined the effects of acute versus chronic PKC activation on I_Ca,L and I_Ca,T in NRVMs. Figure 2, A and B, shows that in control cells, an incubation for 30 min with PMA or ET-1 did not alter I_Ca,L appreciably. While mean I_Ca,L was slightly decreased, this was not significant at any potential. The stimulation of PKC by phorbol esters has discordant effects on I_Ca. Dösemeci et al. (13) reported an increase in this current, whereas Tseng and Boyden (63) saw a decrease and Walsh and Kass (67) observed no change at all. Our experiments show that chronic PMA treatment increased I_Ca, but this increased current almost exactly matched the increase in cell capacitance, such that I_Ca,L density was not significantly changed by PMA (supplemental Fig. 1; note: all supplemental material may be found posted with the online version of this article). Therefore, I_Ca,L expression is not increased in response to PMA treatment in NRVMs. Figure 3 shows that a chronic pretreatment with ET-1 produced a slight increase in both I_Ca,L and I_Ca,T. Neither the reversal potential, activation voltage, nor the voltage for maximum I_Ca was altered by ET-1 for either channel type. The increase in I_Ca,L and I_Ca,T induced by ET-1 was only significant at potentials where the currents are near their largest values. While the blockade of I_Ca,L with nifedipine minimizes the potential contamination of I_Ca,T by I_Ca,L (and >95% of I_Ca activated from E_h = −50 was blocked), such a complication would be most apparent at positive potentials. This was not the case; moreover, the effect of ET-1 on I_Ca,T was significant at −30 mV where there is almost no detectable I_Ca,L. Thus chronic ET-1 pretreatment may slightly enhance both I_Ca types in these myocytes. Although the diverse effects of ET-1 on I_Ca have been reported (12, 28, 30), the experimental conditions of those reports were of acute treatment. The chronic effects of ET-1 reported here are more consistent with the pathophysiological condition of heart failure.

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).

PMA and ET-1 increase I_Na/Ca expression but do not directly alter I_Na/Ca.

We investigated the chronic versus acute effects of PKC activation on the NaCaX activity in NRVMs directly by electrophysiological recording. Figure 4A shows the protocol used to measure I_Na/Ca. Starting at E_h = −90, a step to −45 mV activates and inactivates Na⁺ current. A subsequent step to 0 mV then activates and inactivates I_Ca. Finally, a ramp from +80 to −140 mV was used to assess the Ni-sensitive I_Na/Ca (Fig. 4B). This protocol was repeated in the presence or absence of 5 mM Ni. Figure 4C shows a record of I_Na/Ca after the subtraction of the two traces. An acute exposure of cells to either PMA (1 μM) or ET-1 (0.1 μM) for 10–30 min did not affect I_Na/Ca (Fig. 5, A and B). In contrast, pretreatment (48–72-h exposure) with either PMA or ET-1 significantly increased I_Na/Ca (Fig. 5, C and D). The reversal potential of I_Na/Ca was not altered, assuring that the intracellular Na⁺ and Ca²⁺ concentrations dictated by the pipette solution were the same for control and experimental cells. The ratio of PMA to control or ET-1 to control for I_Na/Ca, indicating the fold change in NaCaX activation, was consistent at nearly all membrane potential (V_m) values evaluated [2.16 ± 0.09 and 1.53 ± 0.07 for PMA (Fig. 5C) and ET-1 (Fig. 5D), respectively]. However, this difference was significant only at V_m values where the current was relatively large. To evaluate inward I_Na/Ca after PMA pretreatment, an additional series of experiments was performed at elevated [Ca²⁺]_i (1 μM) to enhance I_Na/Ca amplitude (supplemental Fig. 2). This procedure shifted the reversal potential to a more positive V_m and confirmed the increased expression of inward I_Na/Ca. Furthermore, we measured the protein level expression for NaCaX and found a twofold increase for both ET-1 (2.26 ± 0.19, P ≤ 0.05) and PMA (2.34 ± 0.27, P ≤ 0.05) (supplemental Fig. 3). Therefore, the effects of PMA or ET-1 treatment on NRVMs mimic changes in NaCaX expression seen in the failing heart.

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.

Pretreatment with PMA and ET-1 decreases I_to.

Chronic heart failure is associated with K⁺ channel misregulation, where decreased K⁺ current prevents repolarization and prolongs AP duration (APD). Therefore, we examined the outward K⁺ currents in response to PMA or ET-1 treatment. Cardiac myocytes were clamped from a holding voltage of −70 to −60 mV for 40 ms and then stimulated with 300-ms pulses from −30 to +120 mV before returning to the E_h. This protocol generated a transient current (I_to), followed by inactivation to a sustained outward level (I_SS) at the end of the pulse (Fig. 6A). An acute exposure to PMA (or ET-1) did not alter either component of this current appreciably (supplemental Fig. 4), a chronic exposure of PMA or ET-1 reduced the I_to component but not the I_SS (Fig. 6, B–D). Thus PMA and ET-1 downregulated the expression of I_to but had no effect on I_SS. Decreased I_to is expected to reduce the amount of early repolarization of the AP and prolong APD. Moreover, the downregulation of I_to is a hallmark of cardiac hypertrophy and heart failure (5, 47) and can contribute to hypertrophy as well (25).

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.

Chronic PMA and ET-1 treatment decreases I_Ks.

Adult rat ventricular myocytes do not normally express the delayed rectifier K⁺ channels, either I_Ks or I_Kr, that are the major repolarizing currents in large mammals. However, in this cultured NRVM model, we detect a slowly activating K⁺ current that resembles classical I_Ks. Figure 7A shows I_Ks assessment in NRVMs by 4-s-long voltage-clamp pulse protocol (55). Both the slowly developing outward current (Fig. 7, C and D) and tail currents at the end of the 4-s pulse (Fig. 7, E and F) were analyzed. An acute application of PMA or ET-1 did not produce substantive changes in I_Ks (supplemental Fig. 5). In contrast, treatment for 48–72 h with PMA or ET-1 decreased I_Ks (Figs. 7, C and D). Raw traces from PMA-treated cells are illustrated in Fig. 7B. Tail K⁺ currents were also depressed by either PMA or ET-1 pretreatment. Figure 7, bottom, shows normalized tail currents from cells pretreated with ET-1 (Fig. 7E) and PMA (Fig. 7F). Consequently, our experiments with NRVMs suggest that there is I_Ks functionally expressed, and chronic exposure to hypertrophic agonists reduces its functional expression, consistent with prolonged APD seen in larger mammals during hypertrophy and heart failure.

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.

I_K1 is borderline reduced by PMA and ET-1 only at higher negative potentials.

Inward rectifying K⁺ current (I_K1) is diminished in adult heart failure (47) contributing to (among other mechanisms) the onset of arrhythmias. The strong I_K1 that is central in stabilizing the resting V_m in ventricular myocytes was assessed in NRVMs using step pulses (duration, 300 ms) from −140 to 0 mV. Raw control traces are shown in Fig. 8A. An acute application of PMA had no effect on I_K1 in NRVMs (supplemental Fig. 6). A chronic pretreatment with ET-1 (and PMA) produced a tendency for reduced I_K1 at the more negative V_m (Fig. 8B), but this trend did not reach statistical significance; during the physiological voltage range (0 to −100 mV), no changes were observed.

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).

APD is increased by PMA and ET-1.

The above effects of 48–72-h PMA or ET-1 exposure to increase predominantly inward currents (I_Ca and I_Na/Ca) and reduce outward currents (I_to and I_Ks) would be expected to prolong APD. Figure 9A shows AP traces with and without PMA and ET-1 pretreatment. Mean data (Fig. 9B) show that a significant prolongation on APD at 80% repolarization (APD₈₀) is observable for both treatments (control APD₈₀ = 252 ± 11.7, PMA APD₈₀ = 342 ± 26.9, and ET-1 APD₈₀ = 279 ± 7.5 ms; P ≤ 0.05). Noticeably, the resting V_m did not change (control, −64.7 ± 2.6; PMA, −63.4 ± 2.5; and ET-1, −61.3 ± 1.4 mV; not significant), which is consistent with the lack of significant changes in I_K1 within the physiological voltage range.

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.

DISCUSSION

Here we characterized PMA and ET-1 effects (both acute and chronic) on the main ionic currents in NRVM APs and excitation-contraction coupling. PMA is a strong direct PKC activator, whereas ET-1 activates G_q-coupled receptors and activates both PKC and 1,4,5-inositol trisphosphate (IP₃) production. Both are known to induce hypertrophy in NRVMs, and these cultured myocytes have been used in hundreds of studies to elucidate signaling pathways and alterations in the expression of key proteins in cardiac myocytes, typically upon chronic agonist activation. However, there has been relatively little characterization of ionic currents in NRVMs, especially in response to hypertrophic agonists. Acute exposures (30 min) sufficient to activate both PKC and IP₃ production did not significantly alter C_m or ion channel activity, suggesting that the acute regulation of these currents by PKC or IP₃-dependent pathways is minimal. On the other hand, a 48–72-h exposure to PMA and ET-1 and washout before measurement resulted in substantial cellular hypertrophy and changes in the current density of many ionic currents. Because the acute effects were minimal, we infer that these chronic changes in current density are the result of the altered expression levels of either the channel proteins themselves or modulators (disproportionate to the degree of cellular hypertrophy). The cellular hypertrophy measured here as cell capacitance agrees with previous reports of the hypertrophic effects of PKC activators and is consistent with a PKC-dependent increase in cell surface area observed optically and by protein expression studies in response to ET-1 treatment of NRVMs (50).

I_Ca,L density was not significantly altered by chronic PMA treatment, but it should be appreciated that the cell surface area was increased by ∼100% (Fig. 1). This means that there was an upregulation of I_Ca,L functional expression that kept pace almost perfectly with the cellular hypertrophy. In contrast, ET-1 pretreatment produced a modest increase in both I_Ca,L and I_Ca,T. It is possible that the exaggerated functional upregulation of I_Ca,L and I_Ca,T is not PKC dependent (since PMA is such a strong PKC activator). ET-1 also causes IP₃ production, which could induce Ca²⁺-dependent changes in transcription. Indeed, IP₃ and Ca²⁺-calmodulin-dependent signaling can lead to an altered transcriptional regulation via calcineurin-NFAT and CaMKII-HDAC pathways (34, 69). In adult hearts, hypertrophy and heart failure have been associated with the enhanced density of I_Ca,T (41) and either unaltered or slightly increased I_Ca,L (7, 38, 46, 47).

It is well established that NaCaX plays a major role in myocyte Ca²⁺ efflux (4). There are reports suggesting an acute PKC-dependent regulation of NaCaX (26, 57), but we did not detect the acute effects of PMA in NRVMs. However, a chronic treatment with ET-1 or PMA increased I_Na/Ca density. Again, this indicates that NaCaX is upregulated above and beyond the overall cellular hypertrophy. Such increases in NaCaX density are also seen in adult myocytes from hypertrophied (37) and failing hearts (46). Furthermore, computer models show how this increased NaCaX can be arrhythmogenic (49). It is possible that ET-1/PMA and hypertrophic signaling increase NaCaX expression, which is beneficial initially but becomes maladaptive in the transition from hypertrophy to heart failure (35).

I_to was dramatically reduced by chronic, but unaltered by acute, PMA and ET-1 exposure. Note that this reduction exceeds the extent of cellular hypertrophy (Fig. 6C), suggesting that there is a net decrease in the rate of I_to functional expression, not simply a dilution of the same number of I_to channels into a bigger cell. Notably I_SS density was unaltered by chronic PMA or ET-1 exposure, consistent with the idea that I_SS expression increases on pace with cellular hypertrophy (so something other than PKC or IP₃ dominates control of this constancy of current density). I_to exhibits rapid activation and inactivation and is responsible for the early repolarization phase of the cardiac AP and, in adult rat and mouse myocytes, is the predominant repolarizing current. I_to reduction is a very common finding in both ventricular hypertrophy and heart failure (3, 6, 40, 47), and reduced I_to can contribute to AP prolongation (especially in rat and mouse). Notably, the early repolarization driven by I_to can also exert critical control over myocyte Ca²⁺ transients by influencing the driving force for I_Ca (53).

While I_Ks is not usually demonstrable in adult rat or mouse hearts, we observed measurable I_Ks in NRVMs, which is reduced by chronic exposure to hypertrophic agonists. Our findings that ET-1 or PMA treatment decrease I_Ks are consistent with the findings of Volders et al. (66) of decreased I_Ks in dogs with hypertrophy induced by atrioventricular blockade. Reduced I_Ks enhances APD and favors the occurrence of early afterdepolarizations. In the theoretical model of ventricular cardiac myocytes implemented by LabHEART, a 50% decrease in I_Ks increased the AP by 12% (APD₉₀ = 202 ms control vs. 181-ms I_Ks reduced) (49). Thus our results with NRVMs concur with theoretical and in vivo animal models of hypertrophy.

Notably, the net effects of PMA and ET-1 were to increase the currents that are predominantly inward currents during the AP (I_Ca and NaCaX) and also decrease repolarizing K⁺ currents (I_to and I_Ks). These are qualitatively like many reports with respect to these currents in adult hypertrophy and heart failure and would all tend to prolong APD (also typical in adult hypertrophy and heart failure). Indeed, we measured AP prolongation in myocytes exposed to PMA or ET-1 for 48–72 h. By and large, PMA and ET-1 produced similar effects with the exception of I_Ca,L, which would be consistent with PKC activation as the most likely candidate in driving most of the altered functional expression. Our results provide a useful survey of overall acute and chronic effects of ET-1 and PMA on NRVM currents, and this should be a valuable complement to the extensive biochemical and morphological work in this cellular model. However, this is only a starting point for more detailed mechanistic studies of each current to better understand the details of the signaling pathways, transcriptional regulation, and specific channel subunits involved in these changes.

NRVMs are a powerful cellular model that has been a workhorse for studies of cardiac cell signaling involved in hypertrophic remodeling of the heart. However, little was known about the fundamental changes in ion currents known to be associated with adult hypertrophy and heart failure in the setting of hypertrophic stimuli in NRVMs. We found little acute effect of PMA or ET-1 exposure on I_Ca, NaCaX, or K⁺ currents in NRVMs. In contrast, chronic exposure to ET-1 (and for the most part PMA) produces changes in current levels that are comparable (at least in direction) with numerous reports from adult hypertrophy and heart failure models. Namely, there was a modest enhancement of I_Ca,L and I_Ca,T, a greater increase in NaCaX, and a reduction in several K⁺ currents (I_to and I_Ks) with a matching increase in APD. While this system and our data here are not a substitute for adult myocyte studies of hypertrophy, this characterization provides a valuable electrophysiological context for signaling studies in NRVMs.

GRANTS

This work was supported by National Heart, Lung, and Blood Institute Grants R37-HL-30077 and P01-HL-80101.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).