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Ca Exchanger by Skeletal Na Channel Isoform Increases Excitation-Contraction Coupling Efficiency in Rabbit Cardiomyocytes
100a
Sunday, February 28, 2016
514-Pos Board B294 CA2D Tides in Cardiomyocytes Under Mechanical Loading Zhong Jian1, Leighton T Izu1, Yi-Je Chen1, Brittani Wood1, Julie Bossuyt1, Kit S Lam2, Ye Chen-Izu1,3. 1 Department of Pharmacology, University of California Davis, Davis, CA, USA, 2Department of Biochemistry and Molecular Medicine, University of California Davis, Davis, CA, USA, 3Department of Biomedical Engineering, University of California, Davis, CA, USA. Abstract Ca2þ signaling is central to cardiac excitation-contraction coupling and extensive studies have revealed various Ca2þ signaling events in ventricular myocytes. In systole, the action potential opens L-type Ca2þ channels and triggers a synchronous release of Ca2þ from SR, which causes a global Ca2þ transient and whole cell contraction. During diastole, Ca2þ is sequestered back into SR and the cytosolic Ca2 concentration is kept low. However, pathological conditions can cause localized and spontaneous Ca2þ release from SR seen as Ca2þ sparks, puffs, embers, and waves. Spontaneous Ca2þ waves can drive Naþ/Ca2þ exchange current to depolarize the membrane potential, manifesting as delayed afterdepolarizations and triggered action potentials that are arrhythmogenic. Here we report a new type of Ca2þ release event that is distinct from the previously known forms, named Ca2þ tide. A Ca2þ tide contains many discernable Ca2þ sparks that occur near synchronously, but does not show a propagating wave front. Ca2þ tides spontaneously arise after the systolic Ca2þ transient in a contracting myocyte and can happen several times during diastole. Interestingly and importantly, the Ca2þ tides are induced by mechanical loading of cardiomyocytes (by embedding cells in our Cell-in-Gel elastic matrix). Moreover, the cardiomyocytes isolated from TAC pressure-overload mouse model exhibited pronounced Ca2þ tides under mechanical loading. In contrast, cardiomyocytes under loadfree conditions did not show any Ca2þ tides. The mechanisms and conditions that give rise to the ‘new kid on the block’ – Ca2þ tides – will be discussed. 515-Pos Board B295 Dyssynchronous CA Removal in Atrial Cardiac Myocytes Felix Hohendanner1, Frank Heinzel1, Lothar Blatter2. 1 Med. Klinik fu¨r Innere Medizin mit Schwerpunkt Kardiologie, Charite´ Berlin - Campus Virchow Klinikum, Berlin, Germany, 2Department of molecular Biophysics and Physiology, Rush University Chicago, Chicago, IL, USA. In heart failure atrial remodeling (AR) leads to impaired contractility, relaxation and atrial fibrillation and contributes to negative clinical outcomes. In myocytes cytosolic Ca-removal after systolic release is primarily achieved by SERCA-pump and Na/Ca-exchange (NCX) activity, allowing atrial relaxation. In atrial myocytes that lack a transverse-tubule system, NCX is located exclusively in the cell periphery where it is exposed to high concentrations of Ca during the cardiac cycle. Previously we found that the relative contribution of NCX to Ca-removal was increased in AR and was associated with a higher propensity of arrhythmogenic Ca waves, i.e. Ca waves triggering action potentials (APs). We hypothesize that dyssynchrony of Ca-removal globally (whole-cell) and locally (peripheral subsarcolemmal (SS) and central (CT) domains) is responsible for impaired relaxation and increased arrhythmogenicity. We used confocal linescan-imaging and Ca sensitive dyes to investigate Ca-removal in remodeled atrial myocytes from a rabbit left ventricular volume-pressure overload systolic HF model. In control atrial myocytes Ca-removal (assessed by the time constant of decay (TAU) of local SS and CT AP-induced Ca transients) is faster in the cell center (CT region) compared to the SS domain (342 5 32 vs. 393 5 31 ms, n = 10 cells). In AR myocytes however, CT TAU was not different from SS TAU (196 5 11 vs. 190 þ 14 ms, n = 10 cells). In addition, in AR cells local dyssynchrony (defined as the standard deviation of TAU divided by mean TAU) in the CT domain was significantly increased compared to control (0.11 5 0.03 vs. 0.04 5 0.01, n = 10 AR and CTRL cells). However despite the increased dyssynchrony, CT Caremoval was 50% and SS Ca-removal 56% faster in AR myocytes as compared to control (n = 10 AR and CTRL cells). In summary, AR myocytes show accelerated but dyssynchronous diastolic Ca-removal which may result in impaired relaxation and increased susceptibility to rhythm disorders. 516-Pos Board B296 Inteplay of Trigger CA2D Waves and CA2D Transient Alternans in Atrial Myocytes Gary Aistrup1, Yohannes Shiferaw2, Rishi Arora1, Georg Gussak1, Soren Grubb3, William Marszalec1, J. Andrew Wasserstrom1. 1 Feinberg Cardiovascular Research Institute, Northwestern University, Feinberg School of Medicine, Chicago, IL, USA, 2Physics, California State University, Northridge, CA, USA, 3Department of Biomedical Sciences, University of Copenhagen, Copenhagen, Denmark.
We previously documented triggered calcium (Ca) wave (TCW) development in atrial myocytes from large animals during rapid pacing. The goal of this study was to investigate the relationship between Ca transient (CaT) alternans and TCWs in atrial myocytes from normal and failing hearts. Ca cycling was measured via confocal microscopy in atrial myocytes isolated from normal and failing canine hearts then loaded with Ca-sensitive fluorescent dyes. Normal atrial myocytes (NAMs; n = 84) or heart failure atrial myocytes (HFAMs; n = 162) were stimulated at increasingly rapid rates leading to TCW development at rates R 3Hz in ~54% of NAMs, but often at lower rates and with greater incidence in ~88% HFAMs. TCW development was contingent on retaining high SR load with diastolic Ca rising 10-20% above that at basal pacing (1Hz) and fractional Ca-release (FCR) being z 0.5 early during rapid pacing epochs. TCWs failed to develop if such FCR was or became 0.5. However, if such FCR was or became [ 0.5, typically few if any TCWs developed or previously developed TCWs ceased, and either larger magnitude TCW-negative CaTs or CaT alternans developed. Overall, CaT alternans developed in ~29% of NAMs and ~24% of HFAMs. Additionally, large TCWs often exhibited a periodicity of about 1 per 4-5 pacing cycles, thus manifesting as large slow-rate CaTs superimposed on small rapid-rate CaTs. In summary, atrial myocytes are predisposed to TCW formation during rapid pacing. Significantly increasing FCR is effective in abrogating TCWs, but often leads to the development of CaT alternans in their stead. Our data suggest that TCWs could cause early afterdepolarizations and, along with Ca alternans, could promote dispersion of repolarization, thus promoting atrial arrhythmogenesis (such as atrial fibrillation) particularly in the setting of heart failure. 517-Pos Board B297 Activation of Reverse Na/Ca Exchanger by Skeletal Na Channel Isoform Increases Excitation-Contraction Coupling Efficiency in Rabbit Cardiomyocytes Natalia S. Torres1, John H.B. Bridge1,2. 1 CVRTI, University of Utah, Salt Lake City, UT, USA, 2Division of Cardiovascular Medicine, University of Utah, Salt Lake City, UT, USA. Our prior work has shown that TTX-sensitive sodium current (INa) affects sarcoplasmic reticular (SR) Ca release. This current activates early reverse NCX. The resulting Ca entry primes the dyadic cleft, which appears to increase Ca channel coupling fidelity (we refer to this as the priming mechanism). The skeletal isoform Nav1.4 is the main TTX-sensitive Na channel expressed in adult rabbit ventricular cardiomyocytes. Here we tested the hypothesis that this is the principal isoform involved in the priming mechanism. We evoked action potentials (AP) in isolated rabbit ventricular cells loaded with fluo-4, and simultaneously recorded Ca transients before and after the application of either relatively low doses of TTX (100 nM) or the specific Nav1.4 inhibitor m-Conotoxin GIIIB (1 mM). 100 nM of TTX will effectively block all skeletal and the neuronal Navs (Nav1.1 is also present with a low level of expression). However this concentration will have a negligible (5%) effect on the cardiac isoform. There was a more prominent AP upstroke velocity reduction in TTX (7351%, n=4) than in GIIB (8151%, n=6, P<0.05). This can be expected given the relative contribution of each Nav isoform to the phase 0 of the AP. However, the effects of either drug on SR Ca release (measured as the transient maximum upstroke velocity) were identical (6950.03% in TTX (n=5) vs. 6953% in GIIIB (n=6), P<0.05). Furthermore, this reduction in SR Ca release is comparable to the value that we obtained previously when total INa was inactivated with a ramp. This result suggests that the Nav skeletal isoform is the main Nav isoform involved in regulating the efficiency of excitation-contraction coupling in rabbit cardiomyocytes. 518-Pos Board B298 Using Action Potential Clamp Data to Determine the Calcium Fluxes and Contributions in Excitation-Contraction Coupling in Vivo in Cardiomyocytes Martin Laasmaa, Marko Vendelin, Rikke Birkedal. Institute of Cybernetics, Tallinn University of Technology, Tallinn, Estonia. Aim: The significance of the different calcium influx pathways like L-type Ca2þ-channels (LTCC), reverse Naþ/Ca2þ-exchange (NCXrev) or Ca2þ-induced Ca2þ-release (CICR) varies with species and the state of a cell. For instance, in case of heart failure, CICR decreases and influx via NCXrev increases. The aim of this study is to develop a method to quantify calcium fluxes in cardiac excitation-contraction coupling under physiological conditions. Methods: We used action potential clamp to measure ILTCC and INCX currents in rainbow trout ventricular myocytes. Fluorescence changes induced by Ca2þ-transients were recorded using 1mM Fluo-4 AM. To measure ILTCC and/or INCX, we used 10mM nifedipine and 1 or 5mM NiCl2 in extracellular solution. Ryanodine receptors and SR calcium ATPase were inhibited by incubating cells in the presence of 10mM ryanodine and 2mM thapsigargin. We used
Sunday, February 28, 2016
514-Pos Board B294 CA2D Tides in Cardiomyocytes Under Mechanical Loading Zhong Jian1, Leighton T Izu1, Yi-Je Chen1, Brittani Wood1, Julie Bossuyt1, Kit S Lam2, Ye Chen-Izu1,3. 1 Department of Pharmacology, University of California Davis, Davis, CA, USA, 2Department of Biochemistry and Molecular Medicine, University of California Davis, Davis, CA, USA, 3Department of Biomedical Engineering, University of California, Davis, CA, USA. Abstract Ca2þ signaling is central to cardiac excitation-contraction coupling and extensive studies have revealed various Ca2þ signaling events in ventricular myocytes. In systole, the action potential opens L-type Ca2þ channels and triggers a synchronous release of Ca2þ from SR, which causes a global Ca2þ transient and whole cell contraction. During diastole, Ca2þ is sequestered back into SR and the cytosolic Ca2 concentration is kept low. However, pathological conditions can cause localized and spontaneous Ca2þ release from SR seen as Ca2þ sparks, puffs, embers, and waves. Spontaneous Ca2þ waves can drive Naþ/Ca2þ exchange current to depolarize the membrane potential, manifesting as delayed afterdepolarizations and triggered action potentials that are arrhythmogenic. Here we report a new type of Ca2þ release event that is distinct from the previously known forms, named Ca2þ tide. A Ca2þ tide contains many discernable Ca2þ sparks that occur near synchronously, but does not show a propagating wave front. Ca2þ tides spontaneously arise after the systolic Ca2þ transient in a contracting myocyte and can happen several times during diastole. Interestingly and importantly, the Ca2þ tides are induced by mechanical loading of cardiomyocytes (by embedding cells in our Cell-in-Gel elastic matrix). Moreover, the cardiomyocytes isolated from TAC pressure-overload mouse model exhibited pronounced Ca2þ tides under mechanical loading. In contrast, cardiomyocytes under loadfree conditions did not show any Ca2þ tides. The mechanisms and conditions that give rise to the ‘new kid on the block’ – Ca2þ tides – will be discussed. 515-Pos Board B295 Dyssynchronous CA Removal in Atrial Cardiac Myocytes Felix Hohendanner1, Frank Heinzel1, Lothar Blatter2. 1 Med. Klinik fu¨r Innere Medizin mit Schwerpunkt Kardiologie, Charite´ Berlin - Campus Virchow Klinikum, Berlin, Germany, 2Department of molecular Biophysics and Physiology, Rush University Chicago, Chicago, IL, USA. In heart failure atrial remodeling (AR) leads to impaired contractility, relaxation and atrial fibrillation and contributes to negative clinical outcomes. In myocytes cytosolic Ca-removal after systolic release is primarily achieved by SERCA-pump and Na/Ca-exchange (NCX) activity, allowing atrial relaxation. In atrial myocytes that lack a transverse-tubule system, NCX is located exclusively in the cell periphery where it is exposed to high concentrations of Ca during the cardiac cycle. Previously we found that the relative contribution of NCX to Ca-removal was increased in AR and was associated with a higher propensity of arrhythmogenic Ca waves, i.e. Ca waves triggering action potentials (APs). We hypothesize that dyssynchrony of Ca-removal globally (whole-cell) and locally (peripheral subsarcolemmal (SS) and central (CT) domains) is responsible for impaired relaxation and increased arrhythmogenicity. We used confocal linescan-imaging and Ca sensitive dyes to investigate Ca-removal in remodeled atrial myocytes from a rabbit left ventricular volume-pressure overload systolic HF model. In control atrial myocytes Ca-removal (assessed by the time constant of decay (TAU) of local SS and CT AP-induced Ca transients) is faster in the cell center (CT region) compared to the SS domain (342 5 32 vs. 393 5 31 ms, n = 10 cells). In AR myocytes however, CT TAU was not different from SS TAU (196 5 11 vs. 190 þ 14 ms, n = 10 cells). In addition, in AR cells local dyssynchrony (defined as the standard deviation of TAU divided by mean TAU) in the CT domain was significantly increased compared to control (0.11 5 0.03 vs. 0.04 5 0.01, n = 10 AR and CTRL cells). However despite the increased dyssynchrony, CT Caremoval was 50% and SS Ca-removal 56% faster in AR myocytes as compared to control (n = 10 AR and CTRL cells). In summary, AR myocytes show accelerated but dyssynchronous diastolic Ca-removal which may result in impaired relaxation and increased susceptibility to rhythm disorders. 516-Pos Board B296 Inteplay of Trigger CA2D Waves and CA2D Transient Alternans in Atrial Myocytes Gary Aistrup1, Yohannes Shiferaw2, Rishi Arora1, Georg Gussak1, Soren Grubb3, William Marszalec1, J. Andrew Wasserstrom1. 1 Feinberg Cardiovascular Research Institute, Northwestern University, Feinberg School of Medicine, Chicago, IL, USA, 2Physics, California State University, Northridge, CA, USA, 3Department of Biomedical Sciences, University of Copenhagen, Copenhagen, Denmark.
We previously documented triggered calcium (Ca) wave (TCW) development in atrial myocytes from large animals during rapid pacing. The goal of this study was to investigate the relationship between Ca transient (CaT) alternans and TCWs in atrial myocytes from normal and failing hearts. Ca cycling was measured via confocal microscopy in atrial myocytes isolated from normal and failing canine hearts then loaded with Ca-sensitive fluorescent dyes. Normal atrial myocytes (NAMs; n = 84) or heart failure atrial myocytes (HFAMs; n = 162) were stimulated at increasingly rapid rates leading to TCW development at rates R 3Hz in ~54% of NAMs, but often at lower rates and with greater incidence in ~88% HFAMs. TCW development was contingent on retaining high SR load with diastolic Ca rising 10-20% above that at basal pacing (1Hz) and fractional Ca-release (FCR) being z 0.5 early during rapid pacing epochs. TCWs failed to develop if such FCR was or became 0.5. However, if such FCR was or became [ 0.5, typically few if any TCWs developed or previously developed TCWs ceased, and either larger magnitude TCW-negative CaTs or CaT alternans developed. Overall, CaT alternans developed in ~29% of NAMs and ~24% of HFAMs. Additionally, large TCWs often exhibited a periodicity of about 1 per 4-5 pacing cycles, thus manifesting as large slow-rate CaTs superimposed on small rapid-rate CaTs. In summary, atrial myocytes are predisposed to TCW formation during rapid pacing. Significantly increasing FCR is effective in abrogating TCWs, but often leads to the development of CaT alternans in their stead. Our data suggest that TCWs could cause early afterdepolarizations and, along with Ca alternans, could promote dispersion of repolarization, thus promoting atrial arrhythmogenesis (such as atrial fibrillation) particularly in the setting of heart failure. 517-Pos Board B297 Activation of Reverse Na/Ca Exchanger by Skeletal Na Channel Isoform Increases Excitation-Contraction Coupling Efficiency in Rabbit Cardiomyocytes Natalia S. Torres1, John H.B. Bridge1,2. 1 CVRTI, University of Utah, Salt Lake City, UT, USA, 2Division of Cardiovascular Medicine, University of Utah, Salt Lake City, UT, USA. Our prior work has shown that TTX-sensitive sodium current (INa) affects sarcoplasmic reticular (SR) Ca release. This current activates early reverse NCX. The resulting Ca entry primes the dyadic cleft, which appears to increase Ca channel coupling fidelity (we refer to this as the priming mechanism). The skeletal isoform Nav1.4 is the main TTX-sensitive Na channel expressed in adult rabbit ventricular cardiomyocytes. Here we tested the hypothesis that this is the principal isoform involved in the priming mechanism. We evoked action potentials (AP) in isolated rabbit ventricular cells loaded with fluo-4, and simultaneously recorded Ca transients before and after the application of either relatively low doses of TTX (100 nM) or the specific Nav1.4 inhibitor m-Conotoxin GIIIB (1 mM). 100 nM of TTX will effectively block all skeletal and the neuronal Navs (Nav1.1 is also present with a low level of expression). However this concentration will have a negligible (5%) effect on the cardiac isoform. There was a more prominent AP upstroke velocity reduction in TTX (7351%, n=4) than in GIIB (8151%, n=6, P<0.05). This can be expected given the relative contribution of each Nav isoform to the phase 0 of the AP. However, the effects of either drug on SR Ca release (measured as the transient maximum upstroke velocity) were identical (6950.03% in TTX (n=5) vs. 6953% in GIIIB (n=6), P<0.05). Furthermore, this reduction in SR Ca release is comparable to the value that we obtained previously when total INa was inactivated with a ramp. This result suggests that the Nav skeletal isoform is the main Nav isoform involved in regulating the efficiency of excitation-contraction coupling in rabbit cardiomyocytes. 518-Pos Board B298 Using Action Potential Clamp Data to Determine the Calcium Fluxes and Contributions in Excitation-Contraction Coupling in Vivo in Cardiomyocytes Martin Laasmaa, Marko Vendelin, Rikke Birkedal. Institute of Cybernetics, Tallinn University of Technology, Tallinn, Estonia. Aim: The significance of the different calcium influx pathways like L-type Ca2þ-channels (LTCC), reverse Naþ/Ca2þ-exchange (NCXrev) or Ca2þ-induced Ca2þ-release (CICR) varies with species and the state of a cell. For instance, in case of heart failure, CICR decreases and influx via NCXrev increases. The aim of this study is to develop a method to quantify calcium fluxes in cardiac excitation-contraction coupling under physiological conditions. Methods: We used action potential clamp to measure ILTCC and INCX currents in rainbow trout ventricular myocytes. Fluorescence changes induced by Ca2þ-transients were recorded using 1mM Fluo-4 AM. To measure ILTCC and/or INCX, we used 10mM nifedipine and 1 or 5mM NiCl2 in extracellular solution. Ryanodine receptors and SR calcium ATPase were inhibited by incubating cells in the presence of 10mM ryanodine and 2mM thapsigargin. We used