Reactive Oxygen Species-Induced Activation of p90 Ribosomal S6 Kinase Prolongs Cardiac Repolarization Through Inhibiting Outward K+ Channel Activity
p90 ribosomal S6 kinase (p90RSK) is activated in cardiomyopathies caused by conditions such as ischemia/reperfusion injury and diabetes mellitus in which prolongation of cardiac repolarization and frequent arrhythmias are common. Molecular mechanisms underlying the electric 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 (Ito,f), slow delayed outward K+ current (IK,slow), and steady-state K+ current (ISS) 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 nontransgenic littermate control mice were similar, as assessed by quantitative reverse transcriptase–polymerase chain reaction, indicating that p90RSK regulates voltage-gated K+ channels through posttranslational 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 2 conserved sites, Ser516 and Ser550. p90RSK expression significantly inhibited Kv4.3- and Kv4.3 and KChIP2-encoded channel activities in HEK 293 cells, whereas p90RSK’s effects were blocked by amino acid mutation(s) at phosphorylation site(s) in Kv4.3. Hydrogen peroxide, a mediator of induced cardiac p90RSK activation in ischemia/reperfusion injury and diabetes mellitus, had effects similar to those of p90RSK on Kv4.3- or Kv4.3- and KChIP2-encoded channels. Fluoromethylketone, a specific p90RSK inhibitor, abolished hydrogen peroxide effects. These findings indicate that p90RSK activation is critical for reactive oxygen species-mediated inhibition of voltage-gated K+ channel activity and leads to prolongation of cardiac repolarization.
- cardiac repolarization
- hydrogen peroxide
- reactive oxygen species
- voltage-gated outward K+ currents
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 (Ito,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 Ito,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 (Ito,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 regard to posttranslational modulation of channel (Ito,f) activity through kinase-mediated phosphorylation. p90 ribosomal S6 kinase (p90RSK) is a serine/threonine kinase that contains 2 functional kinase domains.11 The N-terminal kinase belongs to the AGC group of kinases, which include protein kinase A and protein kinase C. 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 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,16 and hearts from mice with diabetes mellitus induced by streptozotocin.17
p90RSK activation was convincingly linked to cardiac dysfunctions in cardiomyopathies associated with electric remodeling. We tested the hypothesis that p90RSK is an important molecule modulating voltage-gated K+ channel activity and its activation prolongs cardiac repolarization. The electric 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 National Institutes of Health 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 (http://circres.ahajournals.org).
QT Intervals Are Prolonged in p90RSK Transgenic Mice
Surface electrocardiograms from lead II were recorded from the conscious unrestrained nontransgenic littermate control (NLC) and p90RSK-Tg adult (8 to 12 weeks) mice (Figure 1A–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–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 2 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.
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 2 groups (Figure 2B). However, AP amplitudes and resting membrane potentials were not significantly different (Figure 2C–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 through modulation of voltage-gated K+ channels. Outward K+ currents were recorded from myocytes isolated from ventricular-free walls of adult (8 to 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 +60 mV with durations of 4.5 seconds 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, D) compared with those in NLC myocytes (Figure 3A, 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).
Ito,f, IK,slow, and ISS Are Attenuated in p90RSK Transgenic Mouse Ventricular Myocytes
The current decay during 4.5 seconds depolarization in NLC and p90RSK-Tg mouse ventricular myocytes was well described by the sum of 2 exponentials with decay time constants (τdecay) that differed by an order of magnitudes of Ito,f and IK,slow and a noninactivating component, ISS.19,20 Mean±SEM τdecay 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 Ito,f and IK,slow; they were not significantly different (Figure 3G). As reported previously, neither time constant displayed any voltage dependence.19 Mean±SEM Ito,f, IK,slow, and ISS 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 Ito,f, IK,slow, and ISS at +40 mV 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 μmol/L 4-amiopyridine (4-AP) blocking IK,slow118,21 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 reverse transcriptase–polymerase chain reaction 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, or KChIP2 expression between NLC and p90RSK-Tg ventricles (Figure 4). These data indicate that downregulation of outward K+ currents is likely due to posttranslational modulation of K+ channel by p90RSK.
p90RSK Inhibits Kv4.3- and Kv1.5- Rather Than Kv4.2- and Kv2.1-Encoded Channel Activities
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 provided by Dr Jeanne M. Nerbonne’s laboratory at Washington University School of Medicine in St Louis) and green fluorescent protein and with plasmids lacking or having p90RSK sequence. Green fluorescent protein 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-second depolarization revealed that the current decay was well described by the sum of 2 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 did not 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).
p90RSK Phosphorylates Kv4.3
Kv4.3 protein has 2 conserved phosphorylation sequences, RSPS516 and RLRS550 that are located at the C-terminus. To confirm that p90RSK can phosphorylate these 2 serine sites, 4 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 kDa 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 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, 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, I). p90RSK did not affect inactivation time constants and Kv4.3 double mutant-expressed current densities (Supplemental Figure 4E, F, G, H, I). Activation time constants of Kv4.3 single mutant- or double mutant-expressed currents were unaltered by p90RSK (Supplemental Figure 4J). Interestingly, Kv4.3 mutant(s)-expressed currents (Supplemental Figure 4A, C, 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–J). These findings indicate these 2 serine amino acids are also important for channel activity.
Kv4.3 and KChIP2-Encoded 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).
H2O2 Reduces Kv4.3 or Kv4.3 and KChIP2-Encoded Channel Activities, Which Are Blocked by Fluoromethylketone, a p90RSK Specific Inhibitor
Hydrogen peroxide (H2O2) mediates cardiac p90RSK activation in ischemia/reperfusion injury and diabetes mellitus 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 through 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 μmol/L H2O2 for 2 hours (Figure 7B) compared with the 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), whereas inactivation of the 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 μmol/L fmk and 30 μmol/L 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–G). One micron fmk did not affect Kv4.3-encoded channel activity (Figure 7C, E, F, G). fmk also blocked H2O2 effects on Kv4.3- and KChIP2-encoded channel activity (Supplemental Result 4 and Supplemental Figure 6).
Fluoromethylketone Does Not Affect Nontransgenic Littermate Control 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, 3μmol/L fmk did not affect NLC outward K+ currents (Figure 8Ab), but it rescued the K+ current inhibition by p90RSK (Figure 8Ad; mean±SEM). Ipeak, Ito,f, IK,slow, and ISS densities at +40 mV obtained from NLC +3 μmol/L 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). Ipeak, Ito,f, IK,slow, and ISS densities at +40 mV obtained from p90RSK-Tg +3 μmol/L 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 IPeak and Ito,f densities were significantly different (P<0.05) in these 2 groups. Mean±SEM fast and slow decay time constants of the currents recorded from p90RSK-Tg or NLC cells in the presence of 3 μmol/L fmk were not significantly different from those in NLC cells (data not shown). One micron fmk did not 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 2 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 μmol/L fmk for 2 hours was similar to that in NLC cells (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 4 groups were not significantly different (data not shown).
Prolongation of Cardiac Repolarization by Activation of p90RSK
The results presented here reveal that activation of p90RSK inhibited Ito,f, IK,slow, and ISS and led to the prolongation of cardiac repolarization without affecting mRNA levels of Kv subunits. Ito,f decrease was caused by p90RSK phosphorylating Kv4.3 subunit and reduction of IK,slowwas due to p90RSK inhibiting Kv1.5-encoded channel activity. H2O2, a mediator of induced cardiac p90RSK activation in ischemia/reperfusion injury and diabetes mellitus, 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 (Ito,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 posttranslational 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 protein kinase A, protein kinase C, and calcium/calmodulin-dependent kinase II, have been shown to modulate the function of at least one type of K+ channel, including Kv4.2 and Kv4.3.24–29 p90RSK activity is dramatically increased in failing human hearts,15 guinea pig hearts with ischemia or ischemia/reperfusion,16 and hearts from mice with diabetes mellitus induced by streptozotocin,17 in which Ito,f was consistently reported to be downregulated.2–10 It has been reported that p90RSK can phosphorylate Na+/H+ exchanger and troponin I, and its activation provokes cardiac dysfunction as seen in cardiomyopathies caused by conditions such as ischemia/reperfusion injury16 and diabetes mellitus.17 Ito,f is encoded by Kv4.2, Kv4.3, and KChiP2.30 Kv4.3 expresses in both rodent animals30and humans31 and has 2 p90RSK conserved phosphorylation sequences. Our findings indicate that p90RSK is another kinase involved in the downregulation of Ito.f through phosphorylation of Kv4.3. p90RSK accelerates Kv4.3 alone-encoded channel inactivation, but it did not change the inactivation of Kv4.3 and KChIP2-encoded channel or Ito,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 Ito,f complex channel. Kv4.3 with double mutation (Ala516 and Ala550)-expressed currents were significantly decreased, indicating these 2 amino acids are also important for channel activity. p90RSK inhibits IK,slow1(Kv1.5) channel activity, but the mechanism remains to be investigated. Although studies of p90RSK’s effects on IKs and IKr were not performed, analysis of these 2 channel proteins revealed that there is one conserved p90RSK phosphorylation sequence, RRGS27 in IKs channel, and 4 conserved sequences, RRAS283, RQRKRKLS890, RRRT895, and RRLS1137, in the IKrchannel, all of which were reported to be protein kinase A phosphorylation sites,32,33 and phosphorylation of these sites affects both channel activities.32,33 Studies of these 2 channel modulations by p90RSK are warranted.
Our studies showed that p90RSK activation inhibited cardiac Ito,f, IK,slow, and ISS channel activities and led to prolongation of cardiac repolarization in p90RSK-Tg mice at the age of 8 to 12 weeks. This electric 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 to 12 weeks as assessed by gross morphometric, histological, and noninvasive echocardiographic measurements.17 They only exhibited cardiac dysfunction after 6 to 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 through 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 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 p90RSK22 will be very important to address whether p90RSK inhibitor could be a drug for potentially preventing or treating cardiac arrhythmias.
We recorded surface electrocardiograms exhibiting prolongation of QT intervals and observed no ventricular arrhythmias in p90RSK transgenic mice. Further studies including increase in heart rate by a 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 prolongation of cardiac repolarization, are also likely. However, the extensive additional experiments required to address these issues as discussed go beyond the context of the present study.
Sources of Funding
This work is supported by grants from the National Institute of Health to JA (GM-071485 and HL-077789), CY (HL-077789), JT (GM071434-03) and HX (K08 HL088127-01A1). JA and CY are recipients of Established Investigator Awards of the American Heart Association (0740013N and 0740021N).
ZL and JA contributed equally to this work.
Original received October 24, 2007; revision received May 28, 2008; accepted June 24, 2008.
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