Pathophysiologically-Relevant Levels of Endogenous Cardiotonic Steroids Inhibit the Cardiac Na/K...
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Membrane Receptors and Signal Transduction II 1548-Pos Board B278 LKB1 is a Critical Regulator of Early Atrial Growth and Electrophysio- logical Function Grace E. Kim 1 , Jenna L. Ross 2 , Chaoqin Xie 3 , Xiaohong Wu 2 , Monica Palmeri 4 , Vlad G. Zaha 5 , Mohammed Ashraf 2 , Joe G. Akar 5 , Kerry S. Russell 5 , Fadi G. Akar 3 , Lawrence H. Young 1,5 . 1 Cellular & Molecular Physiology, Yale University, New Haven, CT, USA, 2 Cardiovascular Research Center, Yale University School of Medicine, New Haven, CT, USA, 3 Cardiovascular Research Center, Mt. Sinai School of Medicine, New York, NY, USA, 4 Cardiovascular Research Center, Yale University, New Haven, CT, USA, 5 Section of Cardiovascular Medicine, Yale University School of Medicine, New Haven, CT, USA. The liver kinase B1 (LKB1)-AMP activated protein kinase (AMPK) pathway regulates cellular metabolism, polarity, and growth. Although pre- vious studies have documented the maladaptive consequences of LKB1 deletion in the progressive development of atrial fibrillation (AF) and car- diac hypertrophy, its role in chamber-specific pathophysiology remains largely unresolved. Therefore we systematically explored LKB1’s differen- tial effects on structural and ion channel remodeling in mice with LKB1 deletion in cardiomyocytes. Atrial but not ventricular Nav1.5 and Cx40 expression levels were markedly downregulated in 1d neonates, giving rise to a 2-fold prolongation of the P wave on surface ECG. Detailed ex vivo optical action potential (AP) mapping studies revealed presence of marked inter-atrial conduction abnormalities, loss of bi-atrial electrical coupling, and prolonged AP durations in the atria of mice with LKB1 dele- tion. In contrast, ventricular changes developed with a much slower time- course that was consistent with previous studies. AMPK a2 inactivated hearts demonstrated modest overlap in ion channel expression, but retained normal ECG characteristics, cardiac structure and function compared to their LKB1 deleted counterparts. LKB1 deletion triggers a primary atrial electro- physiological remodeling program that precedes structural abnormalities and likely underlies the development of AF. LKB1 may be a novel target for the prevention of AF. 1549-Pos Board B279 Pathophysiologically-Relevant Levels of Endogenous Cardiotonic Steroids Inhibit the Cardiac Na/K ATPase and Activate ERK1/2 Hypertrophic Signaling In Vivo and In Vitro davor pavlovic 1 , safia siddiqui 1 , gurnoor nagi 1 , lucy newbury 1 , william fuller 1 , Anne-Marie L. Seymour 2 , claire sharpe 1 , michael J. shattock 1 , dunja aksentijevic 1 . 1 King’s College London, London, United Kingdom, 2 University of Hull, Hull, United Kingdom. Type-4 cardiorenal syndrome patients develop heart failure as result of kidney failure. While the mechanism of cardiac dysfunction is unknown, elevated levels of circulating endogenous Na/K ATPase inhibitors (NKAi) are detected in patients and animals with kidney failure. We investigated the pathophysio- logical consequences of NKAi elevation in rat myocytes and hearts of animals with chronic kidney disease (CKD). Methods: Intracellular Na concentration ([Nai]) was measured using fluores- cent ratio-imaging microscopy and ERK1/2 phosphorylation by western blot- ting, in adult rat ventricular myocytes. Cardiac Na/K ATPase (NKA) activity was measured using biochemical NKA assays. CKD was induced in rats via 5/6 partial-nephrectomy (PN) or in mice via folic-acid administration (FA). Creatinine serum levels were increased in both models, with kidney dysfunc- tion (and cardiac hypertrophy) being more severe in PN model. Results: Treatment of field-stimulated rat cardiac myocytes with bufalin (10nM) or ouabain (100nM) significantly increased [Nai] by 1.250.3 and 1.950.2mM, respectively, whereas, concentrations of marinobuganenin below 1mM did not. Furthermore, NKAi activated hypertrophic signaling cascades (assessed by ERK1/2 phosphorylation) with a potency order of bufalin (EC50=70nM)>ouabain (EC50=93nM)> marinobuganenin (EC50=6.4mM). Despite small but significant increases in NKA alpha-1/2 expression, NKA ac- tivity in hearts of animals with kidney dysfunction was reduced by 45519% in PN (6 weeks post-PN) and 3459% in FA animals (12 weeks post FA- injection). NKA inhibition was reversed by incubation with a polyclonal anti-Digoxin antibody (binds most structurally-related NKAi), confirming involvement of NKAi. Conclusions: Pathophysiologically-relevant concentrations of bufalin and ouabain, but not marinobufagenin, raise [Nai] and activate hypertrophic signaling pathways in the heart, thus contributing to Na and diastolic Ca over- load and hypertrophy, during CKD. 1550-Pos Board B280 b-Adrenergic Regulation of Cyclic AMP and Ca Current at the T-Tubules and Surface Membrane in Rat Cardiomyocytes Rodolphe Fischmeister, Cristina E. Molina, Youn Kyoung Son. INSERM U769, Cha ˆtenay-Malabry, France. b-Adrenoceptor (b-AR) signalling is severely impaired in heart failure (HF), and this is accompanied by a loss and disorganization of t-tubules. However, how the latter affects the former is unknown. Here, we examined how acute de- tubulation affects the b-AR response of L-type Ca 2þ channel (LTCC) current (I Ca,L ) and membrane cAMP ([cAMP] m ) in adult rat ventricular myocytes (ARVMs) and the contribution of phosphodiesterases PDE3 and PDE4 in this process. ARVMs were infected with an adenovirus encoding a mutant of the olfactory cyclic nucleotide-gated (CNG) channels a subunit to follow [cAMP] m dynamics by recording the associated cationic current. [cAMP] m was also moni- tored by fluorescent imaging using a FRET-based sensor encoding a plasma- membrane targeted cAMP probe (Epac2-camps). Osmotic shock treatment with 1.5M formamide during 15 min induced a loss of t-tubule network as veri- fied by confocal imaging with di-8-ANEPPS staining. Detubulation of ARVMs reduced membrane capacitance by 40% and basal I Ca,L density ~3-fold. Short (15s) applications of isoprenaline (Iso 100nM) increased I Ca,L ~2.5-fold simi- larly in both conditions but had a 30% smaller effect on [cAMP] m in detubulated ARVMs. After Iso washout, I Ca,L and [cAMP] m returned to basal with a ~1.5- fold faster kinetic in detubulated ARVMs than in control, suggesting a faster cAMP degradation by PDEs. The contribution of PDE3 and PDE4 was thus tested using the inhibitors cilostamide (1mM) and Ro20-1724 (10mM), respec- tively. Both inhibitors had a more pronounced effect on Iso response of I Ca,L and [cAMP] m in detubulated ARVMs. Thus, detubulation per se has a profound effect on the b-AR signalling cascade which may contribute to its impairment in HF. This is partly due to a change in the respective contributions of PDE3 and PDE4 to the hydrolysis of cAMP near the membrane and LTCCs. 1551-Pos Board B281 Wnt Signaling Promotes Pacemaker Myocyte Specification of Differenti- ating Cardiac Progenitor Cells Wenbin Liang, Elizabeth H. Kim, Jordan Mak, Eduardo Marba ´n, Hee Cheol Cho. Cardiology, Cedars-Sinai Heart Institute, Los Angeles, CA, USA. Embryonic stem cells (ESCs) can give rise to cardiomyocytes, but mechanisms of cardiac subtype specification to either pacemaker cells or chamber (atrial/ ventricular) cardiomyocytes remain little understood. Canonical Wnt signaling plays a key role for ESC maintenance and becomes inactivated during differ- entiation. We hypothesized that control of Wnt signaling by Dkk1, an endog- enous secreted protein inhibitor of canonical Wnt signaling, may impact cardiac subtype specification during ESC differentiation. Mouse ESCs were treated with activin-A and BMP-4 for 40 hours to initiate cardiac differentia- tion. Flk-1 þ /PdgfR-a þ cardiac progenitors were FACS-purified and seeded as monolayers (day-4). The monolayers were either treated with a saturating level of exogenous Dkk1 (‘‘exo-Dkk1", 150 ng/ml), or cultured in the endoge- nous level of Dkk1 (‘‘endo-Dkk1", 3.2 ng/ml). At day-8, ~50% of cells were positive for cTnT, a pan-cardiac myocyte marker, in both groups. Endo- Dkk1 group exhibited significantly higher levels of cardiac pacemaker cell markers (Tbx18 and Shox2), but lower levels of chamber lineage markers (Nkx2.5, Isl1, Scn5a and Cacna1c, p<0.01). At day-10, endo-Dkk1 group began to beat faster compared to exo-Dkk1 monolayers, and the superior auto- maticity became more accentuated upon further differentiation (161.5511.5 vs. 48.052.9 bpm, endo- vs. exo-Dkk1, week-3). Endo-Dkk1 monolayers yielded more cells with spontaneous intracellular calcium oscillations compared to exo-Dkk1. Single, spontaneously-beating cells isolated from the endo-Dkk1 monolayers were frequently spindle-shaped, exhibited robust HCN4 proteins and I(f) currents, and fired rhythmic action potentials (288551 bpm, n ¼ 5). Analogous results were attained with two other endog- enous Wnt antagonists, Sfrp1 and Sfrp5. Our data demonstrate that the endog- enous Wnt pathway of differentiating ESCs promotes specification of cardiac pacemaker cell, rather than chamber cardiomyocytes. Our findings provide a control point to enrich either pacemaker cell or chamber myocyte population. 1552-Pos Board B282 Modulation of Adrenergic Signalling by Flavonoids in Cardioprotection Aleksey Zholobenko 1 , Eva Gabrielova ´ 2 , Ji rı ´ Ne cas 3 , Martin Modriansky ´ 1 . 1 Medical Chemistry and Biochemistry, Palacky University, Olomouc, Czech Republic, 2 Institute of Molecular and Translational Medicine, Palacky University, Olomouc, Czech Republic, 3 Physiology, Palacky University, Olomouc, Czech Republic. Dietary flavonoids are thought to contribute to the prevention of cardiovascular disease. Recently we showed that 2,3-dehydrosilybin (DHSB) attenuated 304a Monday, February 17, 2014
Transcript of Pathophysiologically-Relevant Levels of Endogenous Cardiotonic Steroids Inhibit the Cardiac Na/K...
304a Monday, February 17, 2014
Membrane Receptors and Signal Transduction II
1548-Pos Board B278LKB1 is a Critical Regulator of Early Atrial Growth and Electrophysio-logical FunctionGrace E. Kim1, Jenna L. Ross2, Chaoqin Xie3, Xiaohong Wu2,Monica Palmeri4, Vlad G. Zaha5, Mohammed Ashraf2, Joe G. Akar5,Kerry S. Russell5, Fadi G. Akar3, Lawrence H. Young1,5.1Cellular & Molecular Physiology, Yale University, New Haven, CT, USA,2Cardiovascular Research Center, Yale University School of Medicine, NewHaven, CT, USA, 3Cardiovascular Research Center, Mt. Sinai School ofMedicine, New York, NY, USA, 4Cardiovascular Research Center, YaleUniversity, New Haven, CT, USA, 5Section of Cardiovascular Medicine,Yale University School of Medicine, New Haven, CT, USA.The liver kinase B1 (LKB1)-AMP activated protein kinase (AMPK)pathway regulates cellular metabolism, polarity, and growth. Although pre-vious studies have documented the maladaptive consequences of LKB1deletion in the progressive development of atrial fibrillation (AF) and car-diac hypertrophy, its role in chamber-specific pathophysiology remainslargely unresolved. Therefore we systematically explored LKB1’s differen-tial effects on structural and ion channel remodeling in mice with LKB1deletion in cardiomyocytes. Atrial but not ventricular Nav1.5 and Cx40expression levels were markedly downregulated in 1d neonates, givingrise to a 2-fold prolongation of the P wave on surface ECG. Detailed exvivo optical action potential (AP) mapping studies revealed presence ofmarked inter-atrial conduction abnormalities, loss of bi-atrial electricalcoupling, and prolonged AP durations in the atria of mice with LKB1 dele-tion. In contrast, ventricular changes developed with a much slower time-course that was consistent with previous studies. AMPK a2 inactivatedhearts demonstrated modest overlap in ion channel expression, but retainednormal ECG characteristics, cardiac structure and function compared to theirLKB1 deleted counterparts. LKB1 deletion triggers a primary atrial electro-physiological remodeling program that precedes structural abnormalities andlikely underlies the development of AF. LKB1 may be a novel target for theprevention of AF.
1549-Pos Board B279Pathophysiologically-Relevant Levels of Endogenous Cardiotonic SteroidsInhibit the Cardiac Na/K ATPase and Activate ERK1/2 HypertrophicSignaling In Vivo and In Vitrodavor pavlovic1, safia siddiqui1, gurnoor nagi1, lucy newbury1,william fuller1, Anne-Marie L. Seymour2, claire sharpe1, michael J. shattock1,dunja aksentijevic1.1King’s College London, London, United Kingdom, 2University of Hull,Hull, United Kingdom.Type-4 cardiorenal syndrome patients develop heart failure as result of kidneyfailure. While the mechanism of cardiac dysfunction is unknown, elevatedlevels of circulating endogenous Na/K ATPase inhibitors (NKAi) are detectedin patients and animals with kidney failure. We investigated the pathophysio-logical consequences of NKAi elevation in rat myocytes and hearts of animalswith chronic kidney disease (CKD).Methods: Intracellular Na concentration ([Nai]) was measured using fluores-cent ratio-imaging microscopy and ERK1/2 phosphorylation by western blot-ting, in adult rat ventricular myocytes. Cardiac Na/K ATPase (NKA) activitywas measured using biochemical NKA assays. CKD was induced in rats via5/6 partial-nephrectomy (PN) or in mice via folic-acid administration (FA).Creatinine serum levels were increased in both models, with kidney dysfunc-tion (and cardiac hypertrophy) being more severe in PN model.Results: Treatment of field-stimulated rat cardiac myocytes with bufalin(10nM) or ouabain (100nM) significantly increased [Nai] by 1.250.3 and1.950.2mM, respectively, whereas, concentrations of marinobuganenin below1mM did not. Furthermore, NKAi activated hypertrophic signaling cascades(assessed by ERK1/2 phosphorylation) with a potency order of bufalin(EC50=70nM)>ouabain (EC50=93nM)> marinobuganenin (EC50=6.4mM).Despite small but significant increases in NKA alpha-1/2 expression, NKA ac-tivity in hearts of animals with kidney dysfunction was reduced by 45519% inPN (6 weeks post-PN) and 3459% in FA animals (12 weeks post FA-injection). NKA inhibition was reversed by incubation with a polyclonalanti-Digoxin antibody (binds most structurally-related NKAi), confirminginvolvement of NKAi.Conclusions: Pathophysiologically-relevant concentrations of bufalin andouabain, but not marinobufagenin, raise [Nai] and activate hypertrophicsignaling pathways in the heart, thus contributing to Na and diastolic Ca over-load and hypertrophy, during CKD.
1550-Pos Board B280b-Adrenergic Regulation of Cyclic AMP and Ca Current at the T-Tubulesand Surface Membrane in Rat CardiomyocytesRodolphe Fischmeister, Cristina E. Molina, Youn Kyoung Son.INSERM U769, Chatenay-Malabry, France.b-Adrenoceptor (b-AR) signalling is severely impaired in heart failure (HF),and this is accompanied by a loss and disorganization of t-tubules. However,how the latter affects the former is unknown. Here, we examined how acute de-tubulation affects the b-AR response of L-type Ca2þ channel (LTCC) current(ICa,L) and membrane cAMP ([cAMP]m) in adult rat ventricular myocytes(ARVMs) and the contribution of phosphodiesterases PDE3 and PDE4 in thisprocess. ARVMs were infected with an adenovirus encoding a mutant of theolfactory cyclic nucleotide-gated (CNG) channels a subunit to follow [cAMP]mdynamics by recording the associated cationic current. [cAMP]mwas also moni-tored by fluorescent imaging using a FRET-based sensor encoding a plasma-membrane targeted cAMP probe (Epac2-camps). Osmotic shock treatmentwith 1.5M formamide during 15 min induced a loss of t-tubule network as veri-fied by confocal imaging with di-8-ANEPPS staining. Detubulation of ARVMsreduced membrane capacitance by 40% and basal ICa,L density ~3-fold. Short(15s) applications of isoprenaline (Iso 100nM) increased ICa,L ~2.5-fold simi-larly in both conditions but had a 30% smaller effect on [cAMP]m in detubulatedARVMs. After Iso washout, ICa,L and [cAMP]m returned to basal with a ~1.5-fold faster kinetic in detubulated ARVMs than in control, suggesting a fastercAMP degradation by PDEs. The contribution of PDE3 and PDE4 was thustested using the inhibitors cilostamide (1mM) and Ro20-1724 (10mM), respec-tively. Both inhibitors had a more pronounced effect on Iso response of ICa,Land [cAMP]m in detubulated ARVMs. Thus, detubulation per se has a profoundeffect on the b-AR signalling cascade whichmay contribute to its impairment inHF. This is partly due to a change in the respective contributions of PDE3 andPDE4 to the hydrolysis of cAMP near the membrane and LTCCs.
1551-Pos Board B281Wnt Signaling Promotes Pacemaker Myocyte Specification of Differenti-ating Cardiac Progenitor CellsWenbin Liang, Elizabeth H. Kim, Jordan Mak, Eduardo Marban, Hee CheolCho.Cardiology, Cedars-Sinai Heart Institute, Los Angeles, CA, USA.Embryonic stem cells (ESCs) can give rise to cardiomyocytes, but mechanismsof cardiac subtype specification to either pacemaker cells or chamber (atrial/ventricular) cardiomyocytes remain little understood. Canonical Wnt signalingplays a key role for ESC maintenance and becomes inactivated during differ-entiation. We hypothesized that control of Wnt signaling by Dkk1, an endog-enous secreted protein inhibitor of canonical Wnt signaling, may impactcardiac subtype specification during ESC differentiation. Mouse ESCs weretreated with activin-A and BMP-4 for 40 hours to initiate cardiac differentia-tion. Flk-1þ/PdgfR-aþ cardiac progenitors were FACS-purified and seededas monolayers (day-4). The monolayers were either treated with a saturatinglevel of exogenous Dkk1 (‘‘exo-Dkk1", 150 ng/ml), or cultured in the endoge-nous level of Dkk1 (‘‘endo-Dkk1", 3.2 ng/ml). At day-8, ~50% of cells werepositive for cTnT, a pan-cardiac myocyte marker, in both groups. Endo-Dkk1 group exhibited significantly higher levels of cardiac pacemaker cellmarkers (Tbx18 and Shox2), but lower levels of chamber lineage markers(Nkx2.5, Isl1, Scn5a and Cacna1c, p<0.01). At day-10, endo-Dkk1 groupbegan to beat faster compared to exo-Dkk1 monolayers, and the superior auto-maticity became more accentuated upon further differentiation (161.5511.5vs. 48.052.9 bpm, endo- vs. exo-Dkk1, week-3). Endo-Dkk1 monolayersyielded more cells with spontaneous intracellular calcium oscillationscompared to exo-Dkk1. Single, spontaneously-beating cells isolated from theendo-Dkk1 monolayers were frequently spindle-shaped, exhibited robustHCN4 proteins and I(f) currents, and fired rhythmic action potentials(288551 bpm, n ¼ 5). Analogous results were attained with two other endog-enous Wnt antagonists, Sfrp1 and Sfrp5. Our data demonstrate that the endog-enous Wnt pathway of differentiating ESCs promotes specification of cardiacpacemaker cell, rather than chamber cardiomyocytes. Our findings provide acontrol point to enrich either pacemaker cell or chamber myocyte population.
1552-Pos Board B282Modulation of Adrenergic Signalling by Flavonoids in CardioprotectionAleksey Zholobenko1, Eva Gabrielova2, Ji�rı Ne�cas3, Martin Modriansky1.1Medical Chemistry and Biochemistry, Palacky University, Olomouc, CzechRepublic, 2Institute of Molecular and Translational Medicine, PalackyUniversity, Olomouc, Czech Republic, 3Physiology, Palacky University,Olomouc, Czech Republic.Dietary flavonoids are thought to contribute to the prevention of cardiovasculardisease. Recently we showed that 2,3-dehydrosilybin (DHSB) attenuated
