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Dynamic Blood Flow Control in Heart
36a
Sunday, February 12, 2017
experiments and the If blocker, ivabradine, to investigate the role of If in pacemaking. Spontaneous APs were recorded from acutely-isolated SAMs from mice, and ~10 s of the AP recording was incorporated into a voltage clamp command. This AP clamp protocol elicited both inward and outward ivabradine-sensitive currents in SAMs. If was defined as the ivabradinesensitive current remaining in the presence of a pharmacological cocktail of sodium, calcium and potassium channel blockers. Measurable inward ivabradine-sensitive current was evident during diastole in 57% of all cells and was generally larger in cells with the most negative diastolic voltages. Although we found that 30 mM ivabradine also blocked both L- and T-type calcium currents in SAMs, the majority of the ivabradine-sensitive inward current in the AP clamp experiments appeared to be If because control experiments showed that the drug cocktail blocked ~85% of the calcium currents. Ivabradine also blocked a substantial outward current during the AP upstroke and repolarization phases. This outward ivabradine-sensitive current likely includes a contribution of If, which results from its relatively slow deactivation. However some of the outward ivabradine-sensitive current may also reflect offtarget block of potassium currents by ivabradine. We are currently assaying the contribution of potassium currents to the outward ivabradine-sensitive current. Our results suggest that If is active not only during diastole, but also throughout the AP upstroke and repolarization phases of the mouse SAM AP. 180-Plat Calcium Activated Chloride Current in Mammalian Ventricular Myocytes Janos Magyar1, Bala´zs Horva´th1, Krisztina Va´czi1, Bence Hegyi1, Mo´nika Go¨nczi2, Beatrix Dienes1, Korne´l Kistama´s1, Tama´s Ba´nya´sz1, Istva´n Baczko´3, Andra´s Varro´3, Gyo¨rgy Sepre´nyi4, La´szlo´ Csernoch1, Pe´ter P. Na´na´si5, Norbert Szentandra´ssy5. 1 Dept. of Physiology, University of Debrecen Faculty of Medicine, Debrecen, Hungary, 2Dept. of Biochemistry and Molecular Biology, University of Debrecen Faculty of Medicine, Debrecen, Hungary, 3Dept. of Pharmacology and Pharmacotherapy, University of Szeged Faculty of Medicine, Szeged, Hungary, 4Dept. of Medical Biology, University of Szeged Faculty of Medicine, Szeged, Hungary, 5Dept. of Physiology, University of Debrecen Faculty of Dentistry, Debrecen, Hungary. Background: Calcium activated Cl current (ICl(Ca)) mediated by TMEM16A and/or Bestrophin-3 may contribute to cardiac arrhythmias. The true profile of ICl(Ca) during an actual ventricular action potential (AP), however, is poorly understood. Our goal was to study the current profile under normal calcium cycling and AP voltage-clamp condition as well as in case of altered intracellular calcium concentration ([Ca2þ]i). The expression of TMEM16A and/or Bestrophin-3 in canine and human left ventricular myocytes was examined. The possible spatial distribution of these proteins and their co-localization with Cav1.2 was also studied. Methods: Whole-cell configuration of the patch-clamp technique and action potential voltage-clamp were used to monitor ICl(Ca), detected as 9-anthracene carboxylic acid (9-AC)-sensitive current. FURA-2-AM dye was used to measure [Ca2þ]i. Expression and cellular localization of Cav1.2, Bestrophin-3 and TMEM16A was analyzed with immunocytochemistry and confocal microscopy. Results: Under AP voltage-clamp conditions the profile of ICl(Ca) contained an early fast outward (1.6250.06 A/F) and a late inward component (0.1650.02 A/F), overlapping early and terminal repolarizations, respectively. Both components were reduced by ryanodine (1.0550.03 A/F; 0.0750.03 A/ F), while fully abolished by BAPTA (0.1350.10 A/F; 0.0850.02 A/F), but not EGTA (1.1750.09 A/F; 0.1350.02 A/F). Setting [Ca2þ]i to the systolic level (1.1 mM) decreased ICl(Ca), while application of Bay K8644, isoproterenol, and faster stimulation rates increased the amplitude of ICl(Ca). Both L-type Ca2þ current and ICl(Ca) were eliminated by nisoldipine. TMEM16A and Bestrophin-3 showed strong co-localization with one another and also with Cav1.2 channels both canine myocytes and human ventricular myocardium. Conclusions: Activation of ICl(Ca) in canine ventricular cells requires calcium entry through neighboring L-type Ca2þ channels and is only augmented by SR Ca2þ-release. Substantial activation of ICl(Ca) requires high Ca2þ in the dyadic clefts which can be effectively buffered by BAPTA, but not EGTA. 181-Plat Properties of New Voltage Sensitive Dyes in Cardiac Field Ndeye Rokhaya Faye1, Sushmitha Raja1, Richard Walton1, Phillipe Pasdois1, Fabien Brette1, Alan Urban2, Alexandre Hentz3, Gihad Dargazanli3, Olivier Bernus1. 1 Electrophysiology and Heart Modelling Institute (IHU-LIRYC), Pessac, France, 2Neuro-Electronics Research Flanders, Imec, Leuven, Belgium, 3 Sanofi, Chilly-Mazarin, Paris, France. These last years, combination of high spatiotemporal resolution techniques with the development of fluorescent voltage sensitive dyes (VSDs) have signif-
icantly improved our understandings of electrophysiological functions of electrically excitable organs (heart, brain). These dyes allow to measure simultaneously membrane potential changes from multiple sites of biological preparations in a non-invasive way. However, conventional dyes have excitation spectra in the blue/green range, which limits the depth of penetration due to high absorbance and photon scattering properties. Therefore, the development of longer wavelength VSDs could have many advantages namely: action potentials recording from tissue’s deeper layers, enhanced voltage sensitivity, and improved spectral properties such that simultaneous monitoring of multiple parameters (i.e. ion transient, pH). The goal of our study is to develop and characterize novel VSDs with large Stoke shifts and near-infrared spectral properties. Three families of VSDs were thus synthesized from two heterocyclic fluorophores and their properties (spectral properties, membrane staining, and dye phototoxicity) assessed in isolated ventricular cells and in Langendorff perfused rat hearts. In addition, special attention is paid to Signal:background ratios and signal kinetics of these new dyes by epifluorescence imaging. Optimal peak emission wavelengths for all dyes was found between 700 nm and 750 nm. The largest observed Stoke shift was found with fluorophore 1 family (about 250 nm). Signal:background ratios ranged from 3% (fluorophore 2) to 7% (fluorophore 3), compared to 5.5% for the usual conventional dye, Di-4-ANEPPS. Rapid signal decay indicative of internalization was observed for one VSD family. However, in other two, signal stability was improved compared to Di-4-ANEPPS. Overall, dyes made with fluorophore 3 show improved voltage sensitivity and potential for applications. 182-Plat Dynamic Blood Flow Control in Heart Guiling Zhao, Humberto Joca, W. Jonathan Lederer. BioMET and Physiology, University of Maryland School of Medicine, Baltimore, MD, USA. Perfusion of the heart by blood is essential for the maintenance of physiological function. Published experiments and investigations by others reveal that the maximum AV O2 difference that can be achieved in heart is always observed between the coronary arteries and the venous outflow at the coronary sinus over the full range of blood flow (~five fold) that is achieved. If so, exquisite matching of blood flow and O2 consumption must exist even at the smallest unit of perfusion. How does such tight control occur? We have examined the hypothesis put forward for the brain to determine if, in principle, it could work in heart. Nelson and colleagues have suggested that electrical activity could be a primary regulator of blood flow in the brain. Increased neuronal activity would lead to an increase in Kþ efflux that would bathe endothelial cells which possess inward rectifier potassium channels (Kir). Activation of Kir could lead to hyperpolarization given the N-shaped Kir current-voltage (IV) relationship that we have observed. In the current study, we examined human microvascular endothelial cells from heart (HMVEC-C) in culture to determine how they responded to elevated [Kþ]o. We found that extracellular increases of Kþ (from 5 mM to 15 mM) cause HMVEC-C hyperpolarization. This Kþ-induced membrane hyperpolarization is dependent on Kir activation as evidenced by its blockade by extracellular Ba2þ. These findings suggest that cardiac electrical activity could contribute to Kþ-dependent hyperpolarization of the endothelial cells that could hyperpolarize the pre-capillary sphincter smooth muscle cells locally and thereby contribute to activity-dependent blood flow control. 183-Plat KChIP2 Serves Multiple Functions in Cardiac Myocytes in Splice Isoform-Dependent Manner Randolph Bettinger1, Rostam Panjshiri1, Drew M. Nassal2, Isabelle Deschenes2, Min Jiang3, Gea-Ny Tseng1. 1 Physiology & Biophysics, Virginia Commonwealth University, Richmond, VA, USA, 2Medicine and Physiology & Biophysics, Case Western University, Cleveland, OH, USA, 3Institute of Medicine Biotechnology, Chinese Academy of Medical Sciences, Beijing, China. K-channel-interacting-proteins (KChIPs) are EF-hand-motif-bearing Ca sensors that serve diverse functions. KChIP2 is the major KChIP expressed in the heart, that can modulate key cardiac channels (Itof, Nav, CaV and IKur) by directly interacting with their pore-forming subunits (Kv4, Nav1.5, Cav1.2, and Kv1.5, respectively) on the cell surface. More recent data suggest that KChIP2 can traffic to nuclei and exert transcriptional activity. Nine KChIP2 splice variants have been identified, that share C-terminal EF-hand-motifs but diverge in N-termini. Based on N-terminal sequences, KChIP2 splice variants can be broadly divided as palmitoylation-capable and palmitoylation-incapable. Questions: (1) Does palmitoylation impact on KChIP2 distribution and function?(2) Is KChIP2 splicing pattern altered in diseased heart? Methods: (1) Tag KChIP2a and 2c
Sunday, February 12, 2017
experiments and the If blocker, ivabradine, to investigate the role of If in pacemaking. Spontaneous APs were recorded from acutely-isolated SAMs from mice, and ~10 s of the AP recording was incorporated into a voltage clamp command. This AP clamp protocol elicited both inward and outward ivabradine-sensitive currents in SAMs. If was defined as the ivabradinesensitive current remaining in the presence of a pharmacological cocktail of sodium, calcium and potassium channel blockers. Measurable inward ivabradine-sensitive current was evident during diastole in 57% of all cells and was generally larger in cells with the most negative diastolic voltages. Although we found that 30 mM ivabradine also blocked both L- and T-type calcium currents in SAMs, the majority of the ivabradine-sensitive inward current in the AP clamp experiments appeared to be If because control experiments showed that the drug cocktail blocked ~85% of the calcium currents. Ivabradine also blocked a substantial outward current during the AP upstroke and repolarization phases. This outward ivabradine-sensitive current likely includes a contribution of If, which results from its relatively slow deactivation. However some of the outward ivabradine-sensitive current may also reflect offtarget block of potassium currents by ivabradine. We are currently assaying the contribution of potassium currents to the outward ivabradine-sensitive current. Our results suggest that If is active not only during diastole, but also throughout the AP upstroke and repolarization phases of the mouse SAM AP. 180-Plat Calcium Activated Chloride Current in Mammalian Ventricular Myocytes Janos Magyar1, Bala´zs Horva´th1, Krisztina Va´czi1, Bence Hegyi1, Mo´nika Go¨nczi2, Beatrix Dienes1, Korne´l Kistama´s1, Tama´s Ba´nya´sz1, Istva´n Baczko´3, Andra´s Varro´3, Gyo¨rgy Sepre´nyi4, La´szlo´ Csernoch1, Pe´ter P. Na´na´si5, Norbert Szentandra´ssy5. 1 Dept. of Physiology, University of Debrecen Faculty of Medicine, Debrecen, Hungary, 2Dept. of Biochemistry and Molecular Biology, University of Debrecen Faculty of Medicine, Debrecen, Hungary, 3Dept. of Pharmacology and Pharmacotherapy, University of Szeged Faculty of Medicine, Szeged, Hungary, 4Dept. of Medical Biology, University of Szeged Faculty of Medicine, Szeged, Hungary, 5Dept. of Physiology, University of Debrecen Faculty of Dentistry, Debrecen, Hungary. Background: Calcium activated Cl current (ICl(Ca)) mediated by TMEM16A and/or Bestrophin-3 may contribute to cardiac arrhythmias. The true profile of ICl(Ca) during an actual ventricular action potential (AP), however, is poorly understood. Our goal was to study the current profile under normal calcium cycling and AP voltage-clamp condition as well as in case of altered intracellular calcium concentration ([Ca2þ]i). The expression of TMEM16A and/or Bestrophin-3 in canine and human left ventricular myocytes was examined. The possible spatial distribution of these proteins and their co-localization with Cav1.2 was also studied. Methods: Whole-cell configuration of the patch-clamp technique and action potential voltage-clamp were used to monitor ICl(Ca), detected as 9-anthracene carboxylic acid (9-AC)-sensitive current. FURA-2-AM dye was used to measure [Ca2þ]i. Expression and cellular localization of Cav1.2, Bestrophin-3 and TMEM16A was analyzed with immunocytochemistry and confocal microscopy. Results: Under AP voltage-clamp conditions the profile of ICl(Ca) contained an early fast outward (1.6250.06 A/F) and a late inward component (0.1650.02 A/F), overlapping early and terminal repolarizations, respectively. Both components were reduced by ryanodine (1.0550.03 A/F; 0.0750.03 A/ F), while fully abolished by BAPTA (0.1350.10 A/F; 0.0850.02 A/F), but not EGTA (1.1750.09 A/F; 0.1350.02 A/F). Setting [Ca2þ]i to the systolic level (1.1 mM) decreased ICl(Ca), while application of Bay K8644, isoproterenol, and faster stimulation rates increased the amplitude of ICl(Ca). Both L-type Ca2þ current and ICl(Ca) were eliminated by nisoldipine. TMEM16A and Bestrophin-3 showed strong co-localization with one another and also with Cav1.2 channels both canine myocytes and human ventricular myocardium. Conclusions: Activation of ICl(Ca) in canine ventricular cells requires calcium entry through neighboring L-type Ca2þ channels and is only augmented by SR Ca2þ-release. Substantial activation of ICl(Ca) requires high Ca2þ in the dyadic clefts which can be effectively buffered by BAPTA, but not EGTA. 181-Plat Properties of New Voltage Sensitive Dyes in Cardiac Field Ndeye Rokhaya Faye1, Sushmitha Raja1, Richard Walton1, Phillipe Pasdois1, Fabien Brette1, Alan Urban2, Alexandre Hentz3, Gihad Dargazanli3, Olivier Bernus1. 1 Electrophysiology and Heart Modelling Institute (IHU-LIRYC), Pessac, France, 2Neuro-Electronics Research Flanders, Imec, Leuven, Belgium, 3 Sanofi, Chilly-Mazarin, Paris, France. These last years, combination of high spatiotemporal resolution techniques with the development of fluorescent voltage sensitive dyes (VSDs) have signif-
icantly improved our understandings of electrophysiological functions of electrically excitable organs (heart, brain). These dyes allow to measure simultaneously membrane potential changes from multiple sites of biological preparations in a non-invasive way. However, conventional dyes have excitation spectra in the blue/green range, which limits the depth of penetration due to high absorbance and photon scattering properties. Therefore, the development of longer wavelength VSDs could have many advantages namely: action potentials recording from tissue’s deeper layers, enhanced voltage sensitivity, and improved spectral properties such that simultaneous monitoring of multiple parameters (i.e. ion transient, pH). The goal of our study is to develop and characterize novel VSDs with large Stoke shifts and near-infrared spectral properties. Three families of VSDs were thus synthesized from two heterocyclic fluorophores and their properties (spectral properties, membrane staining, and dye phototoxicity) assessed in isolated ventricular cells and in Langendorff perfused rat hearts. In addition, special attention is paid to Signal:background ratios and signal kinetics of these new dyes by epifluorescence imaging. Optimal peak emission wavelengths for all dyes was found between 700 nm and 750 nm. The largest observed Stoke shift was found with fluorophore 1 family (about 250 nm). Signal:background ratios ranged from 3% (fluorophore 2) to 7% (fluorophore 3), compared to 5.5% for the usual conventional dye, Di-4-ANEPPS. Rapid signal decay indicative of internalization was observed for one VSD family. However, in other two, signal stability was improved compared to Di-4-ANEPPS. Overall, dyes made with fluorophore 3 show improved voltage sensitivity and potential for applications. 182-Plat Dynamic Blood Flow Control in Heart Guiling Zhao, Humberto Joca, W. Jonathan Lederer. BioMET and Physiology, University of Maryland School of Medicine, Baltimore, MD, USA. Perfusion of the heart by blood is essential for the maintenance of physiological function. Published experiments and investigations by others reveal that the maximum AV O2 difference that can be achieved in heart is always observed between the coronary arteries and the venous outflow at the coronary sinus over the full range of blood flow (~five fold) that is achieved. If so, exquisite matching of blood flow and O2 consumption must exist even at the smallest unit of perfusion. How does such tight control occur? We have examined the hypothesis put forward for the brain to determine if, in principle, it could work in heart. Nelson and colleagues have suggested that electrical activity could be a primary regulator of blood flow in the brain. Increased neuronal activity would lead to an increase in Kþ efflux that would bathe endothelial cells which possess inward rectifier potassium channels (Kir). Activation of Kir could lead to hyperpolarization given the N-shaped Kir current-voltage (IV) relationship that we have observed. In the current study, we examined human microvascular endothelial cells from heart (HMVEC-C) in culture to determine how they responded to elevated [Kþ]o. We found that extracellular increases of Kþ (from 5 mM to 15 mM) cause HMVEC-C hyperpolarization. This Kþ-induced membrane hyperpolarization is dependent on Kir activation as evidenced by its blockade by extracellular Ba2þ. These findings suggest that cardiac electrical activity could contribute to Kþ-dependent hyperpolarization of the endothelial cells that could hyperpolarize the pre-capillary sphincter smooth muscle cells locally and thereby contribute to activity-dependent blood flow control. 183-Plat KChIP2 Serves Multiple Functions in Cardiac Myocytes in Splice Isoform-Dependent Manner Randolph Bettinger1, Rostam Panjshiri1, Drew M. Nassal2, Isabelle Deschenes2, Min Jiang3, Gea-Ny Tseng1. 1 Physiology & Biophysics, Virginia Commonwealth University, Richmond, VA, USA, 2Medicine and Physiology & Biophysics, Case Western University, Cleveland, OH, USA, 3Institute of Medicine Biotechnology, Chinese Academy of Medical Sciences, Beijing, China. K-channel-interacting-proteins (KChIPs) are EF-hand-motif-bearing Ca sensors that serve diverse functions. KChIP2 is the major KChIP expressed in the heart, that can modulate key cardiac channels (Itof, Nav, CaV and IKur) by directly interacting with their pore-forming subunits (Kv4, Nav1.5, Cav1.2, and Kv1.5, respectively) on the cell surface. More recent data suggest that KChIP2 can traffic to nuclei and exert transcriptional activity. Nine KChIP2 splice variants have been identified, that share C-terminal EF-hand-motifs but diverge in N-termini. Based on N-terminal sequences, KChIP2 splice variants can be broadly divided as palmitoylation-capable and palmitoylation-incapable. Questions: (1) Does palmitoylation impact on KChIP2 distribution and function?(2) Is KChIP2 splicing pattern altered in diseased heart? Methods: (1) Tag KChIP2a and 2c