Modulation Of Cardiac Ion Channels By Protein Tyrosine Kinases And Raloxifene

Protein tyrosine kinases (PTKs) are associated with long term cellular processes such as cell growth, differentiation, and apoptosis. Recent studies showed that protein phosphorylation at tyrosine residues modulated ion channels, including Ca²⁺ channel and several types of K⁺ channels, as well as volume-sensitive Cl^- channels. It is believed that protein kinases and phosphatases are the "yin"(negative) and "yang"(positive) of protein phosphorylation and signaling, i.e. protein phosphorylation is dependent on the activity of the kinases and phosphatases. However, it is unclear whether cardiac I_Na is regulated by the interaction of PTKs and PTPs. The present study was designed to determine whether and how native cardiac I_Na would be regulated by the interplay of PTKs and PTPs with specific PTK inhibitors, including the EGFR (epidermal growth factor receptor) kinase inhibitor tyrphostin AG556 (B56), the PDGFR (platelet-derived growth factor receptor) kinase inhibitor tyrphostin AG1295, and the Src-family kinase inhibitor PP2, and also the protein tyrosine Phosphatase (PTP) inhibitor orthovanadate in guinea pig ventricular myocytes using approaches of whole-cell patch voltage clamp, Immunoprecipitation, and Western blot analysis.1. Effects of EGFR kinase inhibitor B56 on sodium current in guinea pig ventricular myocytesMethods: Guinea pig ventricular myocytes were isolated enzymatically and whole cell patch clamp technique was used to record I_Na .Results: B56 inhibited I_Na in a concentration-dependent manner with an EC₅₀ of 7.6 μM. Voltage-dependent I_Na was substantially inhibited by 5 μM B56, and the effect was partially reversed by washout. I_Na (at peak potential of -35 mV) was -23.3±2.0pA/pF for control, -12.4±0.9 pA/pF for 5 μM B56 (n = 10, P < 0.01 vs control), and -20.3±1.9 pA/pF after washout (P < 0.01 vs B56). 5 uM B56 shifted the availability of I_Na to more negative potentials. V_1/2 for availability shifted by 7.8 mV (from -89.9±1.5 mV in control to -97.7±1.1 mV in B56, n = 7, P < 0.01), and the effect was partially reversed by 3.2 mV (to -94.5±1.6 mV) by washout. However, the activation V_1/2 was not significantly changed by B56 (-46.8±0.6 mV for control; -48.5 ±1.0 mV for B56, n = 6, P = NS). I_Na was well fitted to monoexponential functions with time constant of 5.26±0.14 ms (n = 7) in control. However, the recovery slowed down, and was no longer fitted to the monoexponential function after 5 μM B56, and only fitted by bi-exponential functions with rapid and slow time constants of 2.8±0.4 and 219.3±71 ms. The effect was reversed by washout (Ï„: 6.16±0.28 ms, P = NS vs control). B56 at 5 μM had no effect on activation time constant (Ï„_m), but decreased inactivation time constant (Ï„_h) of cardiac I_Na. Ï„_h was decreased from 2.1 ±0.1 ms to 1.8 ± 0.1 ms (n = 7, P<0.01)at-35mV.2. Effects of PDGFR kinase inhibotor Tyrphostin AG1295 and Src-family PTK inhibitor PP2 on sodium current in guinea pig ventricular myocytesMethods: Guinea pig ventricular myocytes were isolated enzymatically and whole cell patch clamp technique was used to record I_Na Tyrphostin AG1295, a selective inhibitor of PDGFR kinase, and PP2, a membrane permeable Src-family PTK inhibitor, were employed to observe whether PDGFR tyrosine kinase or Src-familiy tyrosine kinases would participate in the regulation of I_Na.Results: AG1295 at 100 μM produced no significant inhibition of I_Na (-1.5±2.2%, n = 4, P = NS). PP2 at 5 μM did not significantly affected I_Na in five cells (-1.7 + 0.9%, P = NS).3. Effects of PTP inhibitor orthovanadate (VO₄^3- ) on sodium current in guinea pig ventricular myocytesMethods: Guinea pig ventricular myocytes were isolated enzymatically and whole cell patch clamp technique was used to record I_Na. The effects of the PTP inhibitor VO₄^3- on cardiac I_Na were studied in different set of experiments.Results: 0.3, 1, 3, 5 mM VO₄^3- increased I_Na in a concentration-dependent manner with an EC50 of 1.8 mM. VO₄^3- at 1 mM enhanced voltage-dependent I_Na substantially, and the effect was reversed on washout. I_Na (at -35 mV) was -21.4±3.3 pA/pF during control, -26.2±3.7 pA/pF with 1 mM VO₄^3- (n = 10, P < 0.01 vs control), and -20.1 ±3.0 pA/pF after washout (P < 0.01 vs VO₄^3-). VO₄^3- significantly reduced both Ï„_m and Ï„_h at the potentials of -50 to -10 mV. Ï„_m was decreased from 0.53 ± 0.04ms to 0.41± 0.03 ms, and Ï„_h was decreased from 2.09 ± 0.16ms to 1.65 ± 0.12 ms (n = 7, P < 0.01) at -35 mV. The effects of VO₄^3- on voltage-dependent activation, availability, and recovery of I_Na were also examined. No significant changes in these kinetics were observed with 1 mM VO₄^3-.4. Influence of VO₄^3- on B56 effectsMethods: Guinea pig ventricular myocytes were isolated enzymatically and whole cell patch clamp technique was used to record I_Na.Results: Reduction of cardiac I_Na by B56 was substantially antagonized by the PTP inhibitor VO₄^3-. B56 (5 μM) alone inhibited I_Na by 33.4 ± 2.2% (n = 16, P < 0.01), and the effect was reduced to 8.8 ± 2.3% in the presence of 1 mM VO₄^3- (n = 6, P < 0.01 vs B56 alone). In addition, pretreatment with 1 mM VO₄^3- substantially antagonized the negatively shifted availability V_1/2 of I_Na by B56. V_1/2 of availability (-86.4 ± 1.2 mV) was not significantly changed by 5 μM B56 (-89.7 ± 1.0 mV, n = 6, P = NS) in the co-application of 1 mM VO₄^3- Moreover, the slowed recovery of I_Na from inactivation by B56 was also antagonized by 1 mM VO₄^3-. The recovery was still fitted to monoexponential functions, and differed from that of B56 alone, althoughrecovery time constant was slightly, but significantly increased (to 5.2 ±0.15 from 4.1 ± 0.16 ms, n = 6, P < 0.05) with combination of 5 μM B56 and 1 mM VO₄^3-.5. Effects of EGF on sodium current in guinea pig ventricularmyocytesMethods: Guinea pig ventricular myocytes were isolated enzymatically and whole cell patch clamp technique was used to investigate if EGF would have effect onI_NaResults: EGF, as VO₄^3-, up-regulated I_Na, and accelerated both activation and inactivation of I_Na. The effects of EGF on voltage-dependent activation, availability, and recovery of I_Na were also examined. No significant changes in these kinetics were observed with 100 ng/ml EGF.6. Tyrosine phosphorylation level of cardiac sodium channelsMethods: Tyrosine phosphorylation level of cardiac sodium channels was studied by employing immunoprecipitation and Western blot analysis.Results: VO₄^3- (1 mM) increased tyrosine phosphorylation level of cardiac sodium channels, whereas B56 at 30 μM reduced the phosphorylation level, and the effect was substantially reversed by pretreatment with 1 mM VO₄^3- EGF (100 ng/ml) produced similar effect to that of VO₄^3-.Conclusions1. I_Na was decreased substantially by B56. The result indicates I_Na could be modulated by EGFR kinase.2. Tyrphostin AG1295 and PP2 had no significant effects on I_Na. The results indicate PDGFR kinase and Src PTK may be not involved in the modulation of I_Na.3. PTP inhibitor VO₄^3- increased I_Na significantly. The result indicates I_Na could be modulated by PTP.4. VO₄^3- had influence on the effect of B56. These results indicate that cardiac I_Nais modulated by the interaction of PTKs and PTPs.5. EGF up-regulated I_Na. The result indicates that tyrosine-phosphrylation of cardiac sodium channels increases by activation of EGF receptor.6. Tyrosine phosphorylation levels of cardiac sodium channels were changed correspondingly. These results indicate that cardiac sodium channel protein is phosphorylated by the interaction of tyrosine kinase.The results from the present observation demonstrated the strong evidence that cardiac I_Na in native guinea pig ventricular myocytes was modulated by the interaction of PTPs and EGFR tyrosine kinase. Activation of PTPs would inhibit, whereas EGFR tyrosine kinase favors the activation of cardiac I_Na channels, indicating that both PTPs and EGFR tyrosine kinase regulate electrical excitability of the heart. In summary, the present study has provided the novel evidence that cardiac I_Na is regulated by the interaction of PTKs and PTPs.The selective estrogen receptor modulator(SERM), raloxifene, is widely used in the treatment of postmenopausal osteoporosis, but may also possess cardioprotective properties. It has been reported that raloxifene acutely suppresses ventricular myocyte contractility through inhibition of the L-type calcium current, but it is still not clear that whether raloxifene has effects on other ion channels in heart. The present study was designed to determine whether raloxifene has effects on transient outward potassium current(I_{to 1}), ultra-activated delayed rectifier potassium current(I_kur), rapidly activated delayed rectifier potassium current(I_Kr), slowly activated potassium current(I_Ks) and sodium current by using whole cell patch clamp technique.1. Effects of raloxifene on I_{to 1} and I_Kur in human atrial myocytesMethods: Human atrial myocytes were enzymatically dissociated and whole cell patch clamp was used to record I_{to 1} å’Œ I_KurResults: Raloxifene inhibited I_{to 1} in a concentration-dependent manner with an EC₅₀ of 0.9 μM. Raloxifene at 0.3 to 10 μM suppressed I_{to 1} at test potentials of 0 mV to +60 mV (n = 6, P < 0.05 or P < 0.01 vs control). No significant voltage-dependence was observed. Voltage-dependent I_{to 1} was substantially inhibited by 1 μM raloxifene, and the effect was partially reversed by washout. I_{to 1} (at potential of + 50 mV) was 10.9 ± 1.0 pA/pF for control, 6.1 ± 0.6 pA/pF for 1 μM raloxifene (n = 8, P < 0.01), and 8.2 ± 0.8 pA/pF after washout. The time-dependent recovery of I_{to 1} from inactivation was slowed by 1 μM raloxifene, time constant( Ï„ ) was changed to 105.0 ± 12.4 ms from 66.4 ± 9.5 ms (n = 6, P < 0.05), and the effect was reversed by washout of the drug(x = 69.3 ± 7.5 ms). Raloxifene significantly accelerated I_{to 1} inactivation in a concentration-dependent manner. The inactivation time constant was reduced by 1 μM raloxifene significantly(n = 6, P < 0.01 vs control) at 0 mV to +60 mV. Raloxifeneat 1μM also reduced the time to peak of I_{to 1} at 0 to +60 mV (n = 6, P < 0.01 vs control).Voltage dependence of I_{to 1} activation and inactivation was not affected by the application of raloxifene. Raloxifene inhibited I_Kur in a concentration-dependent manner with an EC₅₀ of 0.7 μM.. The inhibition showed significant voltage-dependence with 1-10 μM, and stronger effect was observed at potentials positive to +10 mV and +60 mV (n =6, P<0.05 or P<0.01 vs 0 mV). Treatment with ICI 182,780 did not affect the inhibitory effects of raloxifene on I_{to 1} and I_Kur2. Effects of 17β-estradiol on I_{to 1} and I_Kur in human atrial myocytesMethods: Human atrial myocytes were enzymatically dissociated and whole cell patch clamp was used to record I_{to 1} å’Œ I_Kur to study whether estrogen had similar inhibitory effects on I_Kur and I_{to 1} as raloxifene.Results: 17β-estradiol decreased I_{to 1} in a concentration-dependent manner with an EC₅₀ of 10.3 μM, E_max was 57.6%, and the effect was reversible. The inactivation time constant of I_{to 1} (at +50 mV) was reduced by application of presence of 10 μM 17β-estradiol (53.1 ± 7.4 ms vs 41.7 ± 7.5 ms of control, n = 5, P < 0.05), but the time to peak of I_{to 1} at +50 mV was not significantly affected (3.7 ± 0.6 ms vs 3.3 ± 0.7 ms of control, n = 5, P = NS) by 17β-estradiol. 17β-estradiol at 10 μM, like raloxifene, had no effect on steady-state activation and inactivation of I_{to 1} . The V_1/2 of activation was 13.2 ±1.9mV in control, and 14.8 ± 2.0 mV for raloxifene (n = 5, P = NS). The V_1/2 for inactivation of I_{to 1} was -24.5 ±1.2 mV and -25.2 ±1.1 mV for control and raloxifene. In addition, the recovery time constant of I_{to 1} was not altered by raloxifene (69.8 ± 12.3 and 71.2 ± 10.5 ms before and after raloxifene, n = 5, P = NS). 1 -30 μM 17β-estradiol had no significant effects on I_Kur Treatment with ICI 182,780 did not affect the inhibitory effects of 17β-estradiol on I_{to 1}3. Effects of raloxifene on cloned hERG channel current (I_HERG) recombinant human cardiac KCNQ1/KCNE1 channel current expressed in HEK 293 cellsMethods: Human cardiac KCNQ1 and KCNE1 genes were cotransfected into HEK293 cells using Lipofectamine 2000^TM, and cell clones stably expressing both genes were selected. Recombinant human cardiac KCNQ1/KCNE1 channel current (I_Ks was recorded using the perforated patch clamp technique. The vector of hERG/pcDNA3 was transfected transiently into HEK293 cells, and cell clones stably expressing the gene were selected. Cloned hERG channel current (I_HERG) was recorded using whole cell patch clamp technique.Results: Raloxifene inhibited I_HERG in a concentration-dependent manner. The step I_HERG (I_HERG,step) and tail current (I_HERG,tail) were substantially suppressed by the application of raloxifene at test potentials of-20 mV to +60 mV (n = 6, P < 0.01 vs control), and the effect was partially reversed by washout. EC50 was 1.1 μM for I_HERG,step, 2.3μM for I_HERG,tail The effect of raloxifene was not use-dependent, and raloxifene did not affect the time course of HERG channel activation (time constants were 238.9 ± 28.9ms in control and 236.4 ± 39.5ms after 1 μM raloxifene, n = 5, P = NS). Raloxifene did not exert effects on channel inactivation, V_1/2 was not significantly changed (-59.1 ± 2.2 mV for control; -62.0 ± 3.1 mV for raloxifene, n = 6, P = NS). Raloxifene inhibited I_Ks in a concentration-dependent manner with an EC50 of 5.1 μM. I_Ks was substantially inhibited at potentials of-20 to +60 mV by the application of raloxifene (n = 6, P < 0.01 vs control). The effect was significantly reversed by washout. 3 μM raloxifene did not shift the activation curve.Conclusions1. Raloxifene inhibited human atrial native I_{to 1} and I_Kur in a concentration-dependent manner, these effects were unaffected by the estrogen receptor antagonist ICI 182,780.2. 17β-estradiol has no significant effect on I_Kur , and shows a weak inhibitory effect on I_{to 1} . Treatment with ICI 182,780 did not affect the inhibitory effects of 17β-estradiol onI_{to 1} .3. Cloned hERG channel current (I_HERG) and recombinant human cardiacKCNQ1/KCNE1 channel current (I_Ks ) expressed in HEK 293 cells were substantially inhibited by raloxifene.The present study demonstrates novel information that raloxifene significantly inhibits human cardiac repolarization potassium currents, including I_to, I_Kur, I_HERG, and I_Ks in a concentration-dependent manner. These effects indicate that raloxifene may have direct effects on heart and provide possible cardiovascular protection.