Reactive Oxygen Species-Induced Activation of p90 Ribosomal S6 Kinase Prolongs Cardiac Repolarization via Inhibiting Outward K⁺ Channel Activity

Abstract

p90 ribosomal S6 kinase (p90RSK) is activated in cardiomyopathies caused by conditions such as ischemia/reperfusion injury (I/R) and diabetes mellitus (DM), in which prolongation of cardiac repolarization and frequent arrhythmias are common. Molecular mechanisms underlying the electrical remodeling in cardiac diseases are largely unknown. In the present study, we determined the role of p90RSK activation in the modulation of voltage-gated K⁺ channel activity determining cardiac repolarization. Mice with increased cardiac p90RSK activity due to transgenic expression of p90RSK (p90RSK-Tg) had prolongation of QT intervals and of ventricular myocyte action potential durations. Fast transient outward K⁺ current (I_to,f), slow delayed outward K⁺ current (I_K,slow) and steady-state K⁺ current (I_SS) were significantly decreased in p90RSK-Tg mouse ventricular myocytes. mRNA levels of Kv4.3, Kv4.2, Kv1.5, Kv2.1 and KChIP2 from ventricles between p90RSK-Tg and non-transgenic littermate control mice were similar, as assessed by quantitative RT-PCR, indicating that p90RSK regulates voltage-gated K⁺ channels through post-translational modification. Kv4.3- and Kv1.5- rather than Kv4.2- and Kv2.1-encoded channels in HEK 293 cells were inhibited by p90RSK. In vitro phosphorylation analysis showed that Kv4.3 was phosphorylated by p90RSK at two conserved sites, Ser516 and Ser550. p90RSK expression significantly inhibited Kv4.3-, and Kv4.3 and KChIP2-encoded channel activities in HEK 293 cells while p90RSK’s effects were blocked by amino acid mutation(s) at phosphorylation site(s) in Kv4.3. Hydrogen peroxide (H₂O₂), a mediator of induced cardiac p90RSK activation in I/R and DM, had effects similar to those of p90RSK on Kv4.3- or Kv4.3 and KChIP2-encoded channels. Fluoromethylketone, a specific p90RSK inhibitor, abolished H₂O₂ effects. These findings indicate that p90RSK activation is critical for reactive oxygen species-mediated inhibition of voltage-gatedK⁺ channel activity and leads to prolongation of cardiac repolarization.

Introduction

Arrhythmia is a common clinical feature in cardiac abnormalities such as coronary artery atherosclerosis, cardiac hypertrophy and failure, and diabetic cardiomyopathy.¹ Downregulation of fast transient outward K⁺ current (I_to,f) has been shown in hypertrophied, failing, ischemic and diabetic hearts, ^2–10 and it is associated with prolongation of cardiac repolarization, prompting speculation that decreased I_to,f may play an important role in the increased incidence of cardiac arrhythmias in these cardiac diseases. Identification of the specific pathways involved in regulation of the channel (I_to,f) activity might lead to the development of innovative therapeutic strategies for preventing the onset of arrhythmias.

Molecular mechanisms underlying cardiac arrhythmias remain poorly understood, particularly in regards to post-translational modulation of channel (I_to,f) activity via kinases mediated phosphorylation. p90 ribosomal S6 kinase (p90RSK) is a serine/threonine kinase which contains two functional kinase domains.¹¹ The N-terminal kinase belongs to the AGC group of kinases, which include protein kinase A (PKA) and protein kinase C (PKC). The N-terminal kinase is responsible for phosphorylating RSK substrates and recognizes the basic consensus motif: (R/K)XRXX(S/T), or RRX(S/T).^{11, 12} The C-terminal kinase belongs to the calcium/calmodulin-dependent kinase (CaMK) group of kinases, and the only known function of the C-terminal kinases is regulation of the N-terminal kinase activity. The C-terminal tail also contains a short docking motif which is responsible for the specific association of p90RSK and ERK1/2.^{11, 13, 14} p90RSK activity is dramatically increased in failing human hearts,¹⁵ guinea pig hearts with ischemia or ischemia/reperfusion injuries (I/R),¹⁶ and hearts from mice with diabetes mellitus (DM) induced by streptozotocin.¹⁷

p90RSK activation was convincingly linked to cardiac dysfunctions in cardiomyopathies associated with the electrical remodeling. We tested the hypothesis that p90RSK is an important molecule modulating voltage-gated K⁺ channel activity and its activation prolongs cardiac repolarization. The electrical remodeling was determined in mice with cardiac-specific expression of p90RSK (p90RSK-Tg), and molecular mechanisms involved in the modulation of voltage-gated K⁺ channel activity by p90RSK were investigated in vitro.

Materials and Methods

Mice were handled in accordance with the NIH Guide for the Care and Use of Laboratory Animals; all protocols were approved by the University of Rochester Animal Study Committee. The methods/protocols used in the present study are detailed in the online data supplement.

Results

QT Intervals are Prolonged in p90RSK Transgenic Mice

Surface electrocardiograms (ECGs) from lead II were recorded from the conscious unrestrained non-transgenic littermate control (NLC) and p90RSK-Tg adult (8–12 weeks) mice (Figure 1A and B). QT interval was determined manually by placing cursors at the beginning of the QRS signal and the end of the T-wave, and it was prolonged in p90RSK-Tg mice (Figure 1B). Mean±SEM QT intervals were significantly longer (P<0.01) in transgenic mice than NLC (63.1±2.3 ms, n=8 versus 51.8±1.4ms, n=9) (Figure 1C). Neither PR intervals nor heart rates (RR intervals) were affected by p90RSK expression (Figure 1C, D and E). Mean±SEM RR intervals were 106.5±2.5 ms (n=9) and 108.9±5.5 ms (n=8), and PR intervals were 34.7±1.3 ms (n=9) and 36.6±1.5 ms (n=8) in NLC and transgenic mice, respectively. When QT intervals were corrected for heart rate, ¹⁸ the differences between these two groups were also significant (P<0.01) (Figure 1C). Mean±SEM QTc intervals were 50.2±0.9 ms (n=9) and 61.2±2.3 ms (n=8) in NLC and p90RSK-Tg mice, respectively.

Figure 1. QT interval prolongation in p90RSK-Tg mice.

ECGs were recorded from NLC (A) and p90RSK-Tg conscious unrestrained mice (B). QT and QTc intervals were significantly prolonged in p90RSK-Tg mice (C) (**P<0.01) compared with those in NLC. PR interval, RR interval and heart rate were not different in non-transgenic and transgenic mice (C, D and E).

Action Potential Durations are Prolonged in p90RSK-Tg Mouse Ventricular Myocytes

Action potentials (APs) recorded from p90RSK-Tg mouse ventricular myocytes were substantially broader than those obtained from NLC cells at physiologically relevant temperature (35°C) with a stimulation frequency of 8 Hz (Figure 2A). Mean±SEM AP durations at 90%, 75%, 50% and 25% repolarization (APD₉₀, APD₇₅, APD₅₀ and APD₂₅) were 12.4.±1.5 ms, 5.2±0.5 ms, 4.0±0.5 ms and 3.0±0.3 ms in NLC (n=5), and 22.9±1.6 ms, 5.6±0.7 ms, 4.0±0.5 ms and 3.0±0.4 ms in p90RSK-Tg ventricular myocytes (n=5), respectively; APD₉₀ were significantly different (P<0.01) between these two groups (Figure 2B). However, AP amplitudes and resting membrane potentials were not significantly different (Figure 2C and D).

Figure 2. Action potential (AP) prolongation in p90RSK-expressing ventricular myocytes.

APs were recorded from mouse ventricular myocyets at 35°C with a stimulation frequency of 8Hz. AP is prolonged in p90RSK-expressing ventricular myocyte compared with that in NLC (A). APD₉₀ is significantly (**P<0.01) prolonged in p90RSK-Tg ventricular cells compared with NLC (B). AP amplitude and resting membrane potential were not significantly different between these two groups (C and D).

Total Outward K⁺ Currents are Attenuated in p90RSK-Tg Mouse Ventricular Myocytes

We next tested the hypothesis that p90RSK activation leads to the prolongation of cardiac repolarization via modulation of voltage-gated K⁺ channels. Outward K⁺ currents were recorded from myocytes isolated from ventricular free walls of adult (8–12 weeks) mice at room temperature (24°C). After establishing whole-cell recording, the currents were recorded by depolarizing to −60 mV and hyperpolarizing −80 mV from a holding potential (HP), −70 mV, to determine cell capacitance. Then, the cells were depolarized from −40 mV to +60mV with durations of 4.5 s or 500 ms and 10 mV interval. Cell capacitances were not different between NLC and p90RSK-Tg ventricular cells (136.0±10.6 pF, n=16 versus 117.2± 7.0 pF, n=15) (Figure 3E). Outward K⁺ currents at all test potentials were lower in cells from transgenic animals (Figure 3B and D) compared with those in NLC myocytes (Figure 3A and C). Mean±SEM peak outward current densities at +40 mV were 64.7±5.8 pA/pF (n=16) and 45.8±3.0 pA/pF (n=15) in NLC and p90RSK-Tg ventricular myocytes, respectively; these values were significantly different (P<0.01) (Figure 3F).

Figure 3. Ventricular outward K⁺ currents are decreased in p90RSK-Tg mice.

Outward K⁺ currents recorded from NLC (A and C) and p90RSK-Tg (B and D) mouse ventricular myocytes were evoked during 4.5-s (A and B) or 500 ms (C and D) depolarizing voltage steps to potentials between −40 and +60 mV from a HP of −70 mV. Each trial was preceded by a brief (20 ms) depolarization to −20 mV to eliminate voltage-gated Na⁺ currents not being blocked by tetrodotoxin. Outward K⁺ currents in NLC (A and C) and transgenic (B and D) myocytes were distinct; peak outward K⁺ current amplitudes are reduced in p90RSK-expressing cells (B and D) compared with those in NLC cells (A and C). The decay phases of K⁺ currents in panels A and B were analyzed to provide amplitudes of I_{to, f}, I_{K, slow} and I_SS in individual cells and normalized to cell capacitance to obtain the current densities. Time constants of fast and slow components were also obtained. Cell capacitance is unchanged by p90RSK expression (E). Mean±SEM current densities of I_peak, I_{to, f}, I_K,slow, and I_SS are significantly decreased (*P<0.05 or **P<0.01) in p90RSK-expressing ventricular myocytes (F). Both slow and fast time constants are unaltered by p90RSK expression (G).

I_to,f, I_K,slow and I_SS are Attenuated in p90RSK Transgenic Mouse Ventricular Myocytes

The current decay during 4.5 s depolarization in NLC and p90RSK-Tg mouse ventricular myocytes was well described by the sum of two exponentials with decay time constants (τ_decay) that differed by an order of magnitudes of I_to,f and I_K,slow and a non-inactivating component, I_SS. ^{19, 20} Mean±SEM τ_decay for fast and slow components of inactivation at +40 mV in NLC (n=16) and p90RSK-Tg ventricular myocytes (n=15) were 123±6 ms and 1328±46 ms, and 134±7 ms and 1449±44 ms, respectively, corresponding to inactivation of I_{to, f} and I_{K, slow}; they were not significantly different (Figure 3G). As reported previously, neither time constant displayed any voltage dependence. ¹⁹ Mean±SEM I_{to, f}, I_{K, slow} and I_SS densities at +40 mV in NLC ventricular cells (n=16) were 32.0±3.8 pA/pF, 21.0±2.0 pA/pF and 9.7±0.7 pA/pF, respectively; mean±SEM densities of I_to,f, I_{K, slow} and I_SS at +40mV were 23.1±2.2 pA/pF, 15.2±1.3 pA/pF and 7.3±0.7 pA/pF in p90RSK-Tg ventricular cells (n=15). They were significantly different (P<0.05) (Figure 3F). 50 µM 4-amiopyridine (4-AP) blocking I_K,slow1 ^{18, 21} was applied to cells, and 4-AP sensitive currents were significantly decreased in p90RSK-Tg ventricular cells compared with NLC (Supplemental Figure 1 and Supplemental Result 1).

mRNAs of Kv4.3, Kv4.2, Kv2.1, Kv1.5, and KChIP2 are Unaltered in p90RSK-Tg Mouse Ventricles

We next determined if K⁺ channel subunit transcripts are affected by p90RSK. SYBR green quantitative RT-PCR was performed on ventricles of NLC (n=5) and p90RSK-Tg mice (n=5) using Kv α and KChIP2 subunit specific primers (Supplemental Table 1). The analysis revealed that there was no significant difference in Kv4.3, Kv4.2, Kv2.1, Kv1.5 and KChIP2 expression between NLC and p90RSK-Tg ventricles (Figure 4). These data indicate that downregulation of outward K⁺ currents is likely due to post-translational modulation of K⁺ channel by p90RSK.

Figure 4. Quantitative RT-PCR revealed mRNA levels of Kv1.5, Kv2.1, Kv4.3, Kv4.2 and KChIP2 in ventricles are not different between NLC and transgenic mice.

The quantity of Kv α and β subunit mRNA was obtained by division of each value by the actin value. The relative expression levels of these α and β subunits were determined.

p90RSK Inhibits Kv4.3- and Kv1.5- rather than Kv4.2- and Kv2.1-encoded Channel Activities

In order to determine which Kv α channel is modulated by p90RSK, HEK 293 cells were transfected with plasmids encoding Kv4.3, Kv4.2, Kv2.1 or Kv1.5 (which were kindly provided by Dr. Jeanne M. Nerbonne’s laboratory at Washington University School of Medicine in St. Louis) and green fluorescent protein (GFP), and with plasmids lacking or having p90RSK sequence. GFP expression allowed us to visualize cells for whole-cell recording. Kv4.3-expressed K⁺ currents were reduced by p90RSK (Figure 5Ab). Kv4.3-encoded channel inactivation was accelerated (Figure 5Ab), and peak current densities were significantly reduced (p<0.05) by p90RSK at +20 to +40 mV (Figure 5Ac). Analysis of the decay phases of outward K⁺ currents evoked during 4.5-s depolarization revealed that the current decay was well described by the sum of two exponentials, and fast and slow inactivation time constants analyzed from Kv4.3- expressed currents with or without p90RSK were voltage independent, and p90RSK significantly accelerated Kv4.3 channel inactivation at +20 to +40 mV (Figure 5Ad). Peak outward Kv1.5 currents were decreased by p90RSK (Figure 5Bb). Kv1.5 current densities were significantly reduced (P<0.01) by p90RSK at −30 to +60 mV (Figure 5Bc). Analysis of Kv4.3 and Kv1.5 K⁺ current activation phases revealed that activation of currents was well described by one exponential. Activation time constants of Kv4.3 or Kv1.5 currents were unchanged at +40 mV by p90RSK (Kv4.3:1.2±0.1 ms, n=11 versus Kv4.3+p90RSK:1.3±0.2 ms, n=11; Kv1.5: 2.1±0.2 ms, n=8 versus Kv1.5+p90RSK: 2.0±0.2 ms, n=9). p90RSK didn’t shift the voltage-dependence of activation of Kv4.3 or Kv1.5 currents (data not shown). Kv4.2 or Kv2.1-expressed currents were unaltered by p90RSK (Supplemental Figure 2 and Supplemental Result 2).

Figure 5. Kv4.3- and Kv1.5-encoded channel activities are inhibited by p90RSK expression.

A. Outward K⁺ currents were evoked during 4.5-s depolarizing voltage steps to potentials between −60 and +60 mV from a HP of −70 mV in HEK 293 cells expressing Kv4.3 (a) or Kv4.3 and p90RSK (b). Kv4.3-expressed K⁺ currents are markedly reduced by p90RSK (b). Kv4.3 current inactivation is accelerated by p90RSK (b). Mean ± SEM Kv4.3 current densities in cells expressing Kv4.3 (open circles; n=11) and Kv4.3 and p90RSK (filled circles; n=11) were plotted as a function of test potentials and they are significantly different (*P<0.05) at voltage of +20 to +40 mV (c). Mean ± SEM fast and slow inactivation time constants (τ values) analyzed from currents recorded from cells expressing Kv4.3 or Kv.4.3 and p90RSK were plotted as a function of test potentials (d). They are voltage independent and they are significantly different at 0 to +40 mV (*P <0.05 or **P<0.01) between these two groups (open circle and square: Kv4.3; filled circle and square: Kv4.3+p90RSK) (d). B. Outward K⁺ currents were evoked during 100-s depolarizing voltage steps to potentials between −40 and +60 mV from a HP of −70 mV in HEK 293 cells expressing Kv1.5 (a) or Kv1.5 and p90RSK (b). Kv1.5-expressed K⁺ currents are markedly reduced (b). Mean ± SEM Kv1.5-(open circles; n=8) and Kv1.5 and p90RSK- expressed current densities (filled circles; n=9) were plotted as a function of test potentials; they are significantly different (**P<0.01) at voltages of −30 to +60 mV.

p90RSK Phosphorylates Kv4.3

Kv4.3 protein has two conserved phosphorylation sequences, RSPS (516) and RLRS (550), are located at the C-terminus. In order to confirm that p90RSK can phosphorylate these two serine sites, four constructs encoding GST-Kv4.3-C-terminus fusion proteins were generated. Results in Supplemental Figure 3 showed that wild type GST-Kv4.3-CT fusion protein at 40 kD was phosphorylated (lane 1), and phosphorylation was largely abolished by double mutations (Ala516 and Ala550) (lane 7). These results indicate that p90RSK phosphorylates Kv4.3 at Ser516 and Ser550. Unexpectedly, phosphorylation intensity of wild type Kv4.3-CT was slightly increased by single amino acid mutation Ala516 (lane 3) or Ala550 (lane 5). These could be due to one mutation enhancing phosphorylation intensity in other site(s). Three same experiments were performed to show the similar results.

p90RSK Inhibits Kv4.3-encoded Channel Activity but Phosphorylation Site Mutation(s) Block Its Effects

We next determined the effects of p90RSK mediated phosphorylation of the Kv4.3 channel. Kv4.3 mutants with single Ala516 or Ala550 or double Ala516 and Ala550 mutations were generated in the rat short form Kv4.3-pcDNA3.1. Coexpression of Kv4.3 mutant(s) with p90RSK in HEK 293 cells was performed, and whole-cell currents were recorded at room temperature (24°C). Single Ala516 mutation abolished the acceleration of Kv4.3 fast component inactivation and decrease in Kv4.3 current density by p90RSK (Supplemental Figure 4A, B, G, H and I); single Ala550 mutation abolished the acceleration of Kv4.3 slow component inactivation and decrease in Kv4.3 current density by p90RSK (Supplemental Figure 4C, D, G, H and I). p90RSK didn’t affect inactivation time constants and Kv4.3 double mutants-expressed current densities (Supplemental Figure 4E, F, G, H and I). Activation time constants of Kv4.3 single mutant- or double mutants-expressed currents were unaltered by p90RSK (Supplemental Figure 4J). Interestingly, Kv4.3 mutant(s)-expressed currents (Supplemental Figure 4A, C and E) were significantly decreased (P<0.01) compared with wild type Kv4.3 (Figure 5Aa). Mean±SEM Kv4.3S516A or Kv4.3S550A or Kv4.3S516AS550A current densities at +40 mV were significantly decreased (P<0.01) compared with wild type Kv4.3 (Supplemental Figure 4G) but activation and inactivation time constants of Kv4.3 mutant(s)-expressed currents at +40 mV were unchanged (Supplemental Figure 4H, I and J).These findings indicate these two serine amino acids are also important for channel activity.

Kv4.3 and KChIP2-enceoded Channel Activity is Reduced but Phosphorylation Site Mutations Block p90RSK’s Effects

As expected, Kv4.3 and KChIP2 expressed currents in HEK 293 cells were reduced by p90RSK expression (Figure 6). Analysis of the decay phases of outward K⁺ currents evoked during 800 ms depolarization revealed that the current decay was well fitted by one exponential. Mean±SEM inactivation time constants of currents in cells expressing Kv4.3 and KChIP2 without (n=14) or with p90RSK (n=14) were 97±7 ms and 100±7 ms at +40 mV, respectively, and they were not significantly different. The peak current densities at −10 to +40 mV were significantly decreased (P<0.05) by p90RSK (Figure 6C). Further studies showed phosphorylation site mutations blocked p90RSK’s effects (Supplemental Result 3 and Supplemental Figure 5).

Figure 6. Kv4.3 and KChIP2-encoded current densities were attenuated by p90RSK.

Outward currents were evoked during 800 ms depolarizing from −60 to +60 mV with +10 mV interval from a HP of −70 mV in HEK-293 cells expressing Kv4.3 and KChIP2 (A), and Kv4.3, KChIP2 and p90RSK (B). Expression of p90RSK markedly reduces Kv4.3 and KChIP2-expressed currents (B). Mean±SEM peak current densities were plotted as a function of test potentials (C). The current densities are decreased at − 10 to +40 mV (*P<0.05; open circle: Kv4.3 and KChIP2, n=14; filled circle: Kv4.3 and KChIP2+p90RSK, n=14).

H₂O₂ Reduces Kv4.3 or Kv4.3 and KChIP2-encoded Channel Activities, Which are Blocked by fmk, a p90RSK Specific Inhibitor

Hydrogen peroxide (H₂O₂) mediates cardiac p90RSK activation in I/R and DM and increases p90RSK activity in different cell types. ²² To test the hypothesis that H₂O₂ modulates Kv4.3- or Kv4.3 and KChIP2-encoded channel activity via p90RSK activation, we recorded currents from HEK 293 cells expressing Kv4.3 or Kv4.3 and KChIP2 with or without H₂O₂ treatment at room temperature (24°C). Kv4.3 channel inactivation was accelerated in cells treated with 30 µM H₂O₂ for 2 hours (Figure 7B) compared with control group (Figure 7A). Peak outward current densities were decreased by H₂O₂ but there was no significant difference (Figure 7E). Analysis of the current decay phases revealed that the inactivation time constant of Kv4.3 fast component was significantly decreased (P<0.05) at +40 mV (Figure 7F) while inactivation of slow component was unchanged in cells treated with H₂O₂ (Figure 7G). As expected, fluoromethylketone (fmk), a specific p90RSK inhibitor,²³ blocked H₂O₂’s effects on Kv4.3 channel inactivation. The currents recorded from HEK 293 cells expressing Kv4.3 pretreated with 1 µM fmk and 30 µM H₂O₂ for 2 hours (Figure 7D) were not different from those in control cells (Figure 7A). Peak current densities and inactivation time constants in the cells with or without H₂O₂ treatment in the presence of fmk were not significantly different (Figure 7E, F and G). One µM fmk did not affect Kv4.3-encoded channel activity (Figure 7C, E, F and G). fmk also blocked H₂O₂ effects on Kv4.3 and KChIP2-encoded channel activity (Supplemental Result 4 and Supplemental Figure 6).

Figure 7. fmk, a specific p90RSK inhibitor, blocks H₂O₂’s effects on Kv4.3-encoded channel.

Outwars K⁺ currents were recorded during 4.5 s depolarizing voltage steps to potentials between −60 and +60 mV from a HP of −70 mV in HEK 293 cells expressing Kv4.3 treated with (B) or without H₂O₂ for 2 hours (A). 30 µM H₂O₂ accelerates the Kv4.3 channel inactivation (B). Peak outward current density is decreased at +40 mV by H₂O₂ (n=7) but there is no significant difference compared with control group (n=17) (E). H₂O₂ significantly accelerates inactivation of fast component (F) (*P<0.05) but inactivation of slow component is not affected (n=7) (G). fmk does not affect Kv4.3 expressed currents (C) in 1 µM concentration (n=18) but it prevents H₂O₂’s effects (n=5) (D). Acceleration of Kv4.3 fast component inactivation by H₂O₂ is abolished by fmk (n=5) (F).

fmk does not Affect NLC Ventricular Outward K⁺ Currents and Action Potential but It Rescues Them Inhibited or Prolonged by p90RSK

We next focused on studies of fmk effects on outward K⁺ currents in NLC and p90RSK-Tg ventricular cells. Ventricular myocytes were incubated with fmk for 2 hours, and outward K⁺ currents and action potentials were recorded at room temperature (24°C). As illustrated in Figure 8A, 3 µM fmk didn’t affect NLC outward K⁺ currents (Figure Ab) but it rescued the K⁺ current inhibition by p90RSK (Figure 8Ad).Mean±SEM I_peak, I_to,f, I_{K, slow} and I_SS densities at +40 mV obtained from NLC+3 µM fmK (n=4) and NLC (n=16) cells were 54.9±12.6, 28.1±7.8 pA/pF, 19.0±4.7 pA/pF and 7.3±0.3 pA/pF, and 64.7±5.8 pA/pF, 32.0±3.8 pA/pF, 21.0±2.0 pA/pF and 9.7±0.7 pA/pF, respectively, and they were not significantly different; mean±SEM I_peak, I_to,f, I_{K, slow} and I_SS densities at +40 mV obtained from p90RSK-Tg+3 µM fmk (n=5) and p90RSK-Tg (n=15) cells were 45.8±3.0 pA/pF, 23.1±2.2 pA/pF, 15.2±1.3 pA/pF and 7.3±0.7pA/pF, and 59.0±5.7 pA/pF, 33.7±4.4 pA/pF, 15.3±2.6 pA/pF and 8.1±1.3, respectively, and I_Peak and I_to,f densities were significantly different (P<0.05) in these two groups. Mean±SEM fast and slow decay time constants of the currents recorded from p90RSK-Tg or NLC cells in the presence of 3 µM fmk were not significantly different from those in NLC cells (data not shown). One µM fmk didn’t affect NLC and p90RSK-Tg ventricular outward K⁺ currents (data not shown). APs recorded from p90RSK-Tg ventricular cells with a stimulation frequency of 1 Hz were substantially broader than those in NLC cells (Figure 8Bc). Mean±SEM APD₉₀, APD₇₅, APD₅₀ and APD₂₅ were 19.0 ±2.6 ms, 9.0±0.9 ms, 4.9±0.3 ms and 3.2±0.3 ms in NLC cells (n=10), and 44.2±7.2 ms, 14.9±2.2 ms, 7.1 ±0.8 ms and 3.7±0.3 ms in p90RSK-Tg cells (n=9), respectively. APD₉₀, APD₇₅, and APD₅₀ in these two groups were significantly different (P<0.01 or P<0.05). As shown in Figure 8Bd, AP recorded from ventricular myocyte expressing p90RSK treated with 3 µM fmk for 2 hours was similar to that in NLC cell (Figure 8Ba) but it was shorter than that in p90RSK–Tg cell (Figure 8Bc). NLC AP was unaltered by fmk (Figure 8Bb). Mean±SEM APD₉₀, APD₇₅, APD₅₀ and APD₂₅ were 11.1±0.7 ms, 8.2±0.7 ms, 6.2 ±1.0 ms and 4.3±0.5 ms in NLC+fmk cells (n=4), and 15.6 ±0.8 ms, 8.8±0.4 ms, 5.8±0.3 ms and 4.0±0.1 ms in p90RSK-Tg+fmk cells (n=6), and they were not significantly different. APD values were not significantly different either between NLC and p90RSK-Tg+fmk groups. AP amplitudes and resting membrane potentials among these four groups were not significantly different (data not shown).

Figure 8. fmk did not affect ventricular outward K⁺ currents and action potential in NLC mice but it rescued them inhibited or prolonged by p90RSK.

A. Outward K⁺ currents were recorded from NLC (a) and p90RSK-Tg ventricular myocyte (c). Three µM fmk does not affect NLC K⁺ currents (b) but it rescues them from p90RSK inhibition (d). B. AP was recorded from NLC (a) and p90RSK-Tg ventricular myocyte (c) with a stimulation frequency of 1Hz. AP is prolonged in p90RSK-expressing ventricular myocyte (c) compared with that in NLC (a). Three µM fmk does not affect NLC AP (b) but it rescues broader p90RSK-Tg AP (b) to normal waveforms (d).

Discussion

Prolongation of Cardiac Repolarization by Activation of p90RSK

The results presented here reveal that activation of p90RSK inhibited I_to,f, I_{K, slow} and I_SS and led to the prolongation of cardiac repolarization without affecting mRNA levels of Kv subunits. I_to,f decrease was caused by p90RSK phosphorylating Kv4.3 subunit and reduction of I_K,slow was due to p90RSK inhibiting Kv1.5-encoded channel activity. H₂O₂, a mediator of induced cardiac p90RSK activation in I/R and DM, had effects similar to p90RSK in the modulation of Kv4.3 or Kv4.3 and KChIP2-encoded channel activity. Importantly, a p90RSK specific inhibitor, fmk, ²³ was able to abolish H₂O₂’s effects on Kv4.3 channel activity and rescued the mouse ventricular K⁺ channel activity inhibited by p90RSK. To our knowledge, this is the first report to document that activation of p90RSK leads to the prolongation of cardiac repolarization by inhibiting voltage-gated outward K⁺ currents.

Downregulation of outward K⁺ currents (I_to,f) has been observed in a variety of cardiac abnormalities associated with prolongation of cardiac repolarization ^2–10 which predisposes to cardiac arrhythmias. Both transcriptional and post-translational modulations of K⁺ channels are able to alter the channel activities. Phosphorylation is a ubiquitous means of modulating protein function; a number of kinases, including PKA, PKC and CamKII have been shown to modulate the function of at least one type of K⁺ channels including Kv4.2 and Kv4.3. ^24–29 p90RSK activity is dramatically increased in failing human hearts,¹⁵ guinea pig hearts with ischemia or I/R,¹⁶ and hearts from mice with DM induced by streptozotocin, ¹⁷ in which I_to,f was consistently reported to be donwregulated.^2–10 It has been reported that p90RSK can phosphorylate Na⁺/H⁺ exchanger and troponin I, and its activation provokes cardiac dysfunction as seen cardiomyopathies caused by conditions such as I/R¹⁶ and DM.¹⁷ I_to,f is encoded by Kv4.2, Kv4.3 and KChiP2.³⁰ Kv4.3 expresses in both rodent animals ³⁰ and humans,³¹ and has two p90RSK conserved phosphorylation sequences. Our findings indicate that p90RSK is another kinase involved in the downregulation of I_to,f via phosphorylation of Kv4.3. p90RSK accelerates Kv4.3 alone-encoded channel inactivation but it didn’t change the inactivation of Kv4.3 and KChIP2-encoded channel or I_to,f. KChIP2 interacts with Kv4.3 (Kv4.2) and decelerates the inactivation of Kv4.3 (Kv4.2) channel, and increases Kv4.3 (Kv4.2) channel expression.³⁰ KChIP2 may play an important role in the p90RSK regulation of I_to,f complex channel. Kv4.3 with double mutations (Ala516 and Ala550)-expressed currents were significantly decreased, indicating these two amino acids are also important for channel activity. p90RSK inhibits I_Kslow1(Kv1.5) channel activity but the mechanism remains to be investigated. Although studies of p90RSK’s effects on I_Ks and I_Kr were not performed, analysis of these two channel proteins revealed that there are one conserved p90RSK phosphorylation sequence, RRGS27 in I_Ks channel, and four conserved sequences, RRAS283, RQRKRKLS890, RRRT895 and RRLS1137, in the I_Kr channel, all of which were reported to be PKA phosphorylation sites,^{32, 33} and phosphorylation of these sites affects both channel activities.^{32, 33} Studies of these two channel modulations by p90RSK are warranted.

Our studies showed that p90RSK activation inhibited cardiac I_to,f, I_{K, slow} and I_SS channel activities and led to prolongation of cardiac repolarization in p90RSK-Tg mice at the age of 8–12 weeks. This electrical remodeling could be caused by cardiac structural abnormality induced by activation of p90RSK. However, cell capacitances representative of cell sizes were not different between NLC and p90RSK-Tg groups at this age. In addition, previous studies showed that p90RSK-Tg mice did not have any phenotypic changes to suggest cardiac hypertrophy and dysfunction at the age of 8–12 weeks, as assessed by gross morphometric, histological and noninvasive echocardiographic measurements.¹⁷ They only exhibited cardiac dysfunction after 6–8 months of age.¹⁷ All these findings support that p90RSK directly affects voltage-gated outward K⁺ channel activity.

Novelty and Potential Significance

p90RSK has been shown to be activated in different cardiac diseases to potentially modulate cardiac function.^{15–17, 22, 34} Our study is first to show that activation of p90RSK prolongs the QT-interval via inhibiting outward K⁺ channel activity. Our results are relevant to the understanding of the molecular determinants of prolongation of cardiac repolarization predisposing to cardiac arrhythmias and potentially to the development of new therapeutic approaches. The present study demonstrates that targeted inhibition of p90RSK and subsequent prevention of Kv channel activity reduction in response to agonists such as H₂O₂ produced in different types of cardiac abnormalities. Further studies of perturbing p90RSK signaling in animal models by the specific inhibitor or dominant negative p90RSK ²² will be very important to address whether p90RSK inhibitor could be a drug for potentially preventing or treating cardiac arrhythmias.

Potential Limitations

We recorded surface ECGs exhibiting prolongation of QT-intervals and observed no ventricular arrhythmias in p90RSK transgenic mice. Further studies including increase in heart rate by β-receptor agonist or intracardiac pacing may induce ventricular tachycardia or fibrillation in these transgenic mice and provide a useful animal model for antiarrhythmic drug selection. In this study, we focused on voltage-gated outward K⁺ currents altered by p90RSK but effects of p90RSK on other ionic currents including inward-rectifier K⁺ currents, L-type Ca²⁺ currents and Na⁺ currents participating in activation of p90RSK-induced the prolongation of cardiac repolarization are also likely. However, the extensive additional experiments required to address these issues as discussed above go beyond the context of the present study.