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Regulation of L-type Calcium Channel Activity by P21 activated kinase-1 in Guinea-pig Sino-atrial Node Pacemaker Cells
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ABSTRACTS / Journal of Molecular and Cellular Cardiology 40 (2006) 920 – 1015
179. Concentration-dependent effects of PKA inhibitor H-89 on I Ca I to and I K1 in isolated rat ventricular myocytes N. Bracken, C. Pearman, W. Kent, M. El-Kadri, G. Hart. Hussain M School of Clinical Sciences, University of Liverpool, UK H-89 is a protein kinase inhibitor that has a Ki of 50 nM for PKA, 500 nM for PKG and 5 –50 uM for PKC and CamKII (Hidaka & Kobayashi (1992)Ann Rev Toxicol Pharmacol32: 377 –97). However, these values were determined using the purified catalytic subunits of these kinases in vitro. In this study we examined whether these differences in the selectivity were transferable to intact myocytes. Basal I Ca, I K1 and I to were recorded using perforated patch clamp with amphotericin B and internal and external solutions that were appropriate for each of these currents. H-89 inhibited all three current types in a concentrationdependent manner. However, the IC50 was 5.0 AM for I Ca, 30.0 AM for I to and 37.7 for I K1. The effect of H-89 on I Ca was consistent with inhibition of PKA-mediated phosphorylation that sustains basal I Ca. The higher Ki values for I to and I K1 suggests that these effects may be mediated by kinases other than PKA. Data demonstrate that the effective concentrations of H-89 in intact myocytes are ¨100-fold higher than those obtained using the purified catalytic subunits of the kinases in vitro, perhaps due to incomplete equilibration to the interior of the myocyte or non-specific binding to other intracellular proteins. Furthermore, any selectivity of H-89 for PKA is limited due to effects on other targets and must therefore be qualified in the context of the concentrations used. doi:10.1016/j.yjmcc.2006.03.194
180. c-Src-mediated phosphorylation of cardiac L-type Ca2+ channel regulates I Ca through multiple binding sites E. Dubuis a, N. Rockliffe a, M. Hussain b, M. Boyett c, S. Wray d, D. Gawler a. a The Physiological Laboratory, University of Liverpool, UK. b School of Clinical Sciences, University of Liverpool, UK. c The Department of Medicine, University of Manchester, UK. d The School of Biomedical Sciences, University of Leeds, UK Src has been proposed to activate L-type calcium channel by binding to the C-terminus of the a1c subunit. The II – III linker region of this subunit also contains a novel consensus sequence for Src binding. We examined whether this site is a functional Src interaction site and investigated the effect of displacing Src from this region on I Ca. In vitro binding assays were performed to map a1c subunit interaction sites using isolated cardiac myocytes. I Ca were recorded in myocytes loaded with the Src inhibitor PP1 and/or peptides with amino acid sequence corresponding to the hypothesized Src docking site. Co-immunoprecipitation and pull-down studies were undertaken to identify proteins co-complexing with a1c. Peptides corresponding to the II –III linker region and C-
terminal tail of a1c, but not scrambled sequence peptide controls, were found to inhibit Src’s SH3 domain binding to the channel and significantly reduced I Ca. The II – III linker region peptide increased the voltage sensitivity of inactivation whereas the C-terminal tail region peptide decreased the voltage sensitivity of activation when compared to scrambled sequence peptides. PP1-pre-treatment of myocytes also reduced I Ca, decreased the V1/2 for channel inactivation and abolished any further effect on currents by Src binding peptides. The tyrosine kinase PYK2 was found to coassociate with Src and the channel, but PP1-pre-treatment reduced this co-association. We concluded that Src differentially modulates I Ca through two binding sites. PYK2 is able to co-complex with catalytically active Src when bound to this region of the channel. These kinases may synergistically regulate channel activity. doi:10.1016/j.yjmcc.2006.03.195
181. Regulation of L-type Calcium Channel Activity by P21 activated kinase-1 in Guinea-pig Sino-atrial Node Pacemaker Cells Ming Lei a, Yunbo Ke b, Mark S. Brodie b, R. John Solaro b. a Division of Cardiovascular and Endocrine Sciences, University of Manchester, Manchester M13 9XX. b Department of Physiology and Biophysics, University of Illinois at Chicago, Chicago, IL6061, Chicago, IL 60612, USA Modulation of ion channel activity is a fundamental mechanism for heart function including cardiac pacemaker activity. Pathways regulating the phosphorylation of ion channel in cardiac cells are relatively well understood; however, the counterbalance mechanism by dephosphorylation of these proteins remains unclear. Here we report a novel role of P21 activated kinase-1-mediated signaling in regulation of L-type Ca2+ channel activity in guinea-pig sinoatrial node (SAN) pacemaker cells. Endogenous Pak1 is abundantly expressed in SAN cells. Moreover, expression of a constitutively active Pak1 showed a distinct pattern that differs from that of the endogenous Pak1. After 12 hrs infection with AdLacz (control virus) and AdPak1, basal L-type Ca2+ current (ICaL) in cells in AdPak1-infected group and the group infected with the control virus was statistically indistinguishable. However, the ICa.L current in Ad-Lacz group was significantly enhanced in the presence of 100 nM ISO, the peak current at 0 mV increased from 15.0 T 3.0 to 28.0 T 1.3 pApF1(P < 0.01, n = 7) and current amplitude increased by 57 T 6%. However, the effect of ISOstimulation on ICaL in the Ad-Pak1 group was much less effective (100 nM induced an increase in peak current at 0 mV by 13 T 3% from 13.0 T 1.0 pApF1 in the absence of ISO to 16.0 T 1.4 pApF1 in the presence 100 nM ISO, P > 0.05, n = 6). Our immuno-precipitation study indicated that Pak1, PP2A form a complex, which suggests that Pak1 may regulate the activities of L-type Ca2+ channel through phosphatase PP2A in SAN pacemaker cells.
ABSTRACTS / Journal of Molecular and Cellular Cardiology 40 (2006) 920 – 1015
Acknowledgments The study was supported by NIH (RJS) and The Wellcome Trust (ML). doi:10.1016/j.yjmcc.2006.03.196
182. Validation of a new time and frequency domain measure of heart rate variability Terence B. Willard a, Rod A. Little b, Mahesh Nirmalan c. a South Manchester University Hospitals. b MRC Trauma Group, University of Manchester (Retired). c Critical Care Unit, Manchester Royal Infirmary, Manchester, UK The hypothesis that critical illness and injury is associated with reduced heart rate variability (HRV) has been confirmed in many clinical settings. However standard measures of HRV using Fast Fourier Transform have not always provided consistent results and the physiological significance of the specific peaks in the different frequency bands remains inconclusive. Newer methods based on nonlinear mathematics too remain controversial. In particular, the method of approximate entropy (ApEn) may give misleading results. In this study a novel normalized entropy measure ‘‘L’’ was developed for quantifying HRV. ‘‘L’’ provides a linear scale ranging between 0 –1 and was validated using several synthetic time series of increasing complexity and ECG data obtained in an animal model of hypovolaemic shock. Standard 3-lead ECG signals obtained from 29 anaesthetized/ventilated pigs were sampled at 1 kHz and processed using standard time/frequency domain measures of variability and ‘‘L’’ value at baseline, shock phase and post resuscitation. Shock phase was associated with a reversible reduction in ‘‘L’’ value (Repeated measures ANOVA, P < 0.05). The ‘‘L’’ measure was shown to be valid in the time and frequency domains for theoretical and experimental data sets. The study confirms the inconsistencies of ApEn and in particular, the method, when used with conventional parameters (r and m) was of limited value in quantifying different grades of complexity at the three phases of the experiment. Power spectral analysis, after correction for changes in heart rate, showed a significant reduction in total amplitude and high frequency amplitude at the same phases as ‘‘L’’ value. doi:10.1016/j.yjmcc.2006.03.197
183. A novel model of heart rate variability based on stochastic ion channel kinetics in the sino-atrial node (SA node) Mahesh Nirmalan a, Terry B. Willard b, Mahesan Niranjan c. a Critical Care Unit Manchester Royal Infirmary. b Medical Physics, South Manchester University Hospital. c Department of Computer Sciences, University of Sheffield, UK We propose a simple model of heart rate variability (HRV) where HRV is considered to be an inherent property of the heart,
987
subject to external modulation by autonomic nerves. The model is based on the stochastic properties of ion channels involved in the generation of pace maker potentials in the SA node. During stochastic channel opening the number of open channels during each phase of the pace-maker potential would vary based on an appropriate probability distribution resulting in a beat-to-beat variation in the duration of the pace maker potentials. The nature of this probability distribution under a given set of conditions would inevitably lead to beat-to-beat variations in RR intervals (summated electrical activity). If the open probability of the channels is increased (or decreased) markedly due to an external modulator [sympathetic (or parasympathetic) stimulation] channel opening would necessarily follow a deterministic model and under these conditions the number of open channels is correlated between cycles. We model correlated opening as: Nk ¼ ð1 aÞN þ aNk1 Where N k and N k-1 refer to the number of open channels in cycles K and K-1 respectively; a represents the degree of correlation. The model implies that as the degree of correlation increases [i.e the more marked sympathetic stimulation (or failure) is, the more regular the heart rate would be. The model is unique in that it acknowledges HRV as an inherent property of the heart and provides a mechanistic explanation for reduced HRV in states of sympathetic stimulation or failure. Further studies are necessary to validate the above model. doi:10.1016/j.yjmcc.2006.03.198
184. Is the electrophysiological actions of psychotrop drugs responsible for their cardiac side effects? Valeria Kecskeme´ti a, J. Magyar b, T. Ba´nya´sz b, N. Szentandra´ssy b, P. Pacher a, P.P. Na´na´si b. Department of Pharmacology and Pharmacotherapy, Semmelweis University, Budapest. b Department of Physiology, University Medical School of Debrecen, Hungary The cardiac side effects (brady- or, tachycardia, ECG alterations) of psychotrop (antidepressant, antipsychotic) drugs can be due to their effects on cardiac ion channels. Aim: the actions of fluoxetine (F), a selective serotonin reuptake inhibitor antidepressant and risperidone (R), an atypical (DA2/5-HT2 antagonist) antipsychotic drug on isolated canine ventricular myocytes and guinea-pig papillary muscles were analysed using microelectrode and whole cell clamp techniques. Results: at concentrations of 0.5 – 50 AM, F exhibited depressant effects on contraction and both on Ca2+ and Na+dependent electrophysiological parameters of cardiac preparations and on cardiac Ca2+ current, without modifying the K+ currents. R (0.1 – 10 AM) caused a concentration-dependent lengthening of action potential duration (APD) in both preparations and it blocked concentration-dependently the rapid component of the delayed rectifier K+ current (IKr) The other K+ currents (IK1 and Ito) were not significantly modified. Conclusion: the inhibition of cardiac Ca2+ and Na+ currents by F and the depression of IKr current by R may explain the
ABSTRACTS / Journal of Molecular and Cellular Cardiology 40 (2006) 920 – 1015
179. Concentration-dependent effects of PKA inhibitor H-89 on I Ca I to and I K1 in isolated rat ventricular myocytes N. Bracken, C. Pearman, W. Kent, M. El-Kadri, G. Hart. Hussain M School of Clinical Sciences, University of Liverpool, UK H-89 is a protein kinase inhibitor that has a Ki of 50 nM for PKA, 500 nM for PKG and 5 –50 uM for PKC and CamKII (Hidaka & Kobayashi (1992)Ann Rev Toxicol Pharmacol32: 377 –97). However, these values were determined using the purified catalytic subunits of these kinases in vitro. In this study we examined whether these differences in the selectivity were transferable to intact myocytes. Basal I Ca, I K1 and I to were recorded using perforated patch clamp with amphotericin B and internal and external solutions that were appropriate for each of these currents. H-89 inhibited all three current types in a concentrationdependent manner. However, the IC50 was 5.0 AM for I Ca, 30.0 AM for I to and 37.7 for I K1. The effect of H-89 on I Ca was consistent with inhibition of PKA-mediated phosphorylation that sustains basal I Ca. The higher Ki values for I to and I K1 suggests that these effects may be mediated by kinases other than PKA. Data demonstrate that the effective concentrations of H-89 in intact myocytes are ¨100-fold higher than those obtained using the purified catalytic subunits of the kinases in vitro, perhaps due to incomplete equilibration to the interior of the myocyte or non-specific binding to other intracellular proteins. Furthermore, any selectivity of H-89 for PKA is limited due to effects on other targets and must therefore be qualified in the context of the concentrations used. doi:10.1016/j.yjmcc.2006.03.194
180. c-Src-mediated phosphorylation of cardiac L-type Ca2+ channel regulates I Ca through multiple binding sites E. Dubuis a, N. Rockliffe a, M. Hussain b, M. Boyett c, S. Wray d, D. Gawler a. a The Physiological Laboratory, University of Liverpool, UK. b School of Clinical Sciences, University of Liverpool, UK. c The Department of Medicine, University of Manchester, UK. d The School of Biomedical Sciences, University of Leeds, UK Src has been proposed to activate L-type calcium channel by binding to the C-terminus of the a1c subunit. The II – III linker region of this subunit also contains a novel consensus sequence for Src binding. We examined whether this site is a functional Src interaction site and investigated the effect of displacing Src from this region on I Ca. In vitro binding assays were performed to map a1c subunit interaction sites using isolated cardiac myocytes. I Ca were recorded in myocytes loaded with the Src inhibitor PP1 and/or peptides with amino acid sequence corresponding to the hypothesized Src docking site. Co-immunoprecipitation and pull-down studies were undertaken to identify proteins co-complexing with a1c. Peptides corresponding to the II –III linker region and C-
terminal tail of a1c, but not scrambled sequence peptide controls, were found to inhibit Src’s SH3 domain binding to the channel and significantly reduced I Ca. The II – III linker region peptide increased the voltage sensitivity of inactivation whereas the C-terminal tail region peptide decreased the voltage sensitivity of activation when compared to scrambled sequence peptides. PP1-pre-treatment of myocytes also reduced I Ca, decreased the V1/2 for channel inactivation and abolished any further effect on currents by Src binding peptides. The tyrosine kinase PYK2 was found to coassociate with Src and the channel, but PP1-pre-treatment reduced this co-association. We concluded that Src differentially modulates I Ca through two binding sites. PYK2 is able to co-complex with catalytically active Src when bound to this region of the channel. These kinases may synergistically regulate channel activity. doi:10.1016/j.yjmcc.2006.03.195
181. Regulation of L-type Calcium Channel Activity by P21 activated kinase-1 in Guinea-pig Sino-atrial Node Pacemaker Cells Ming Lei a, Yunbo Ke b, Mark S. Brodie b, R. John Solaro b. a Division of Cardiovascular and Endocrine Sciences, University of Manchester, Manchester M13 9XX. b Department of Physiology and Biophysics, University of Illinois at Chicago, Chicago, IL6061, Chicago, IL 60612, USA Modulation of ion channel activity is a fundamental mechanism for heart function including cardiac pacemaker activity. Pathways regulating the phosphorylation of ion channel in cardiac cells are relatively well understood; however, the counterbalance mechanism by dephosphorylation of these proteins remains unclear. Here we report a novel role of P21 activated kinase-1-mediated signaling in regulation of L-type Ca2+ channel activity in guinea-pig sinoatrial node (SAN) pacemaker cells. Endogenous Pak1 is abundantly expressed in SAN cells. Moreover, expression of a constitutively active Pak1 showed a distinct pattern that differs from that of the endogenous Pak1. After 12 hrs infection with AdLacz (control virus) and AdPak1, basal L-type Ca2+ current (ICaL) in cells in AdPak1-infected group and the group infected with the control virus was statistically indistinguishable. However, the ICa.L current in Ad-Lacz group was significantly enhanced in the presence of 100 nM ISO, the peak current at 0 mV increased from 15.0 T 3.0 to 28.0 T 1.3 pApF1(P < 0.01, n = 7) and current amplitude increased by 57 T 6%. However, the effect of ISOstimulation on ICaL in the Ad-Pak1 group was much less effective (100 nM induced an increase in peak current at 0 mV by 13 T 3% from 13.0 T 1.0 pApF1 in the absence of ISO to 16.0 T 1.4 pApF1 in the presence 100 nM ISO, P > 0.05, n = 6). Our immuno-precipitation study indicated that Pak1, PP2A form a complex, which suggests that Pak1 may regulate the activities of L-type Ca2+ channel through phosphatase PP2A in SAN pacemaker cells.
ABSTRACTS / Journal of Molecular and Cellular Cardiology 40 (2006) 920 – 1015
Acknowledgments The study was supported by NIH (RJS) and The Wellcome Trust (ML). doi:10.1016/j.yjmcc.2006.03.196
182. Validation of a new time and frequency domain measure of heart rate variability Terence B. Willard a, Rod A. Little b, Mahesh Nirmalan c. a South Manchester University Hospitals. b MRC Trauma Group, University of Manchester (Retired). c Critical Care Unit, Manchester Royal Infirmary, Manchester, UK The hypothesis that critical illness and injury is associated with reduced heart rate variability (HRV) has been confirmed in many clinical settings. However standard measures of HRV using Fast Fourier Transform have not always provided consistent results and the physiological significance of the specific peaks in the different frequency bands remains inconclusive. Newer methods based on nonlinear mathematics too remain controversial. In particular, the method of approximate entropy (ApEn) may give misleading results. In this study a novel normalized entropy measure ‘‘L’’ was developed for quantifying HRV. ‘‘L’’ provides a linear scale ranging between 0 –1 and was validated using several synthetic time series of increasing complexity and ECG data obtained in an animal model of hypovolaemic shock. Standard 3-lead ECG signals obtained from 29 anaesthetized/ventilated pigs were sampled at 1 kHz and processed using standard time/frequency domain measures of variability and ‘‘L’’ value at baseline, shock phase and post resuscitation. Shock phase was associated with a reversible reduction in ‘‘L’’ value (Repeated measures ANOVA, P < 0.05). The ‘‘L’’ measure was shown to be valid in the time and frequency domains for theoretical and experimental data sets. The study confirms the inconsistencies of ApEn and in particular, the method, when used with conventional parameters (r and m) was of limited value in quantifying different grades of complexity at the three phases of the experiment. Power spectral analysis, after correction for changes in heart rate, showed a significant reduction in total amplitude and high frequency amplitude at the same phases as ‘‘L’’ value. doi:10.1016/j.yjmcc.2006.03.197
183. A novel model of heart rate variability based on stochastic ion channel kinetics in the sino-atrial node (SA node) Mahesh Nirmalan a, Terry B. Willard b, Mahesan Niranjan c. a Critical Care Unit Manchester Royal Infirmary. b Medical Physics, South Manchester University Hospital. c Department of Computer Sciences, University of Sheffield, UK We propose a simple model of heart rate variability (HRV) where HRV is considered to be an inherent property of the heart,
987
subject to external modulation by autonomic nerves. The model is based on the stochastic properties of ion channels involved in the generation of pace maker potentials in the SA node. During stochastic channel opening the number of open channels during each phase of the pace-maker potential would vary based on an appropriate probability distribution resulting in a beat-to-beat variation in the duration of the pace maker potentials. The nature of this probability distribution under a given set of conditions would inevitably lead to beat-to-beat variations in RR intervals (summated electrical activity). If the open probability of the channels is increased (or decreased) markedly due to an external modulator [sympathetic (or parasympathetic) stimulation] channel opening would necessarily follow a deterministic model and under these conditions the number of open channels is correlated between cycles. We model correlated opening as: Nk ¼ ð1 aÞN þ aNk1 Where N k and N k-1 refer to the number of open channels in cycles K and K-1 respectively; a represents the degree of correlation. The model implies that as the degree of correlation increases [i.e the more marked sympathetic stimulation (or failure) is, the more regular the heart rate would be. The model is unique in that it acknowledges HRV as an inherent property of the heart and provides a mechanistic explanation for reduced HRV in states of sympathetic stimulation or failure. Further studies are necessary to validate the above model. doi:10.1016/j.yjmcc.2006.03.198
184. Is the electrophysiological actions of psychotrop drugs responsible for their cardiac side effects? Valeria Kecskeme´ti a, J. Magyar b, T. Ba´nya´sz b, N. Szentandra´ssy b, P. Pacher a, P.P. Na´na´si b. Department of Pharmacology and Pharmacotherapy, Semmelweis University, Budapest. b Department of Physiology, University Medical School of Debrecen, Hungary The cardiac side effects (brady- or, tachycardia, ECG alterations) of psychotrop (antidepressant, antipsychotic) drugs can be due to their effects on cardiac ion channels. Aim: the actions of fluoxetine (F), a selective serotonin reuptake inhibitor antidepressant and risperidone (R), an atypical (DA2/5-HT2 antagonist) antipsychotic drug on isolated canine ventricular myocytes and guinea-pig papillary muscles were analysed using microelectrode and whole cell clamp techniques. Results: at concentrations of 0.5 – 50 AM, F exhibited depressant effects on contraction and both on Ca2+ and Na+dependent electrophysiological parameters of cardiac preparations and on cardiac Ca2+ current, without modifying the K+ currents. R (0.1 – 10 AM) caused a concentration-dependent lengthening of action potential duration (APD) in both preparations and it blocked concentration-dependently the rapid component of the delayed rectifier K+ current (IKr) The other K+ currents (IK1 and Ito) were not significantly modified. Conclusion: the inhibition of cardiac Ca2+ and Na+ currents by F and the depression of IKr current by R may explain the