DOI: 10.1161/CIRCULATIONAHA.115.017980
Late INa in the Heart: Physiology, Pathology, and Pathways
Running title: Makielski et al.; Late INa regulatory pathways Jonathan C. Makielski, MD; John W. Kyle, PhD
Dept of Medicine, Division of Cardiovascular Medicine, University of Wiscon Wisconsinnsi sinn- M Madison, adis ad ison is on, on Madison, WI
Address A dd dress for Correspondence: Corrres Co rr spond pond nden nce ce:: Jonathan Jo Jona o athan C. Makielski, M kieelskii, MD Ma Depa part pa rtme rt ment me nt of Medicine, Meddici Me cine ne, e Division Divi visi vi sion si on of Cardiovascular Card Ca rdio rd iova io vasc va scul sc ular ar M edic ed icin ic inee in Department Medicine University of Wisconsin- Madison 600 Highland Ave H4/5 Madison, WI 53792 Tel: 608-262-2075 Fax: 608-263-0405 E-mail: [email protected]
Journal Subject Codes: Atherosclerosis:[145] Genetically altered mice, Atherosclerosis:[138] Cell signaling/signal transduction, Basic science research:[132] Arrhythmias - basic studies, Atherosclerosis:[148] Heart failure - basic studies, Myocardial biology:[104] Structure
Key words: Editorial, long QT syndrome, calcium/calmodulin-dependent protein kinase II, arrhythmia, sodium current, Scn5a
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DOI: 10.1161/CIRCULATIONAHA.115.017980
In this issue of Circulation, Hund and colleagues1 make an important contribution to understanding the physiological and pathophysiological roles of late sodium current (INa) in the heart, with a focus on a key pathway regulating late INa amplitude. They conducted well designed and detailed studies with two new genetically engineered mouse lines: one a S571A mouse that ablates phosphorylation by Ca2+/calmodulin-dependent kinase II (CaMKII)2 selectively at serine 571 in the cardiac Na channel pore-forming protein Scn5a, and the other a S571E mouse that mimics phosphorylation at serine 571. Serine 571 was shown previously to be a target for phosphorylation by CaMKII and this phosphorylation enhanced late INa2. The present studies in “knock-in” mice expressing either S571A or S571E have distinct advantages over other earlier studies tudies in heterologous expression systems including cultured myocyte models because bec ecau ec ause au se they the hey y allow the study of whole animal and organ phenotypes, as well as the study of cellular and molecular mole leecu cula larr bi la biop biophysical o hyysi s cal properties in a more nativ native ve en environment. nvironment. What th these hese new in vivo studies reveal eveeal is that des despite espite i e tthe he ex exte extensive tens te nsiive ns ive nnetwork etw twoorkk of CaMKII tw CaM MKIII targets, targ ta a getts, phosphorylation phoosp spho horyylati ho tion ti on ooff S S571 571 selectively regulates ele l ct ctively re reg gula late a es late laatee INa aand nd inn pa pparticular rticculaar eenhanced nhan an ncedd llate atee INa inn failing fail ilin il ng hea heart. art. t. Pe ak INa iiss th thee large larg la rgee inward rg inwa in ward ard current curre urrre rent nt fflowing lowi lo wing ing m mainly ainl ai nly th nl thro through roug ro ugh gh th thee ca card cardiac rdia rd iacc Na+ cchannel ia hann ha nnel nn el pore por oree Peak formed by Scn5a, which is part of a larger sodium channel macromolecular complex. Members of this macromolecular complex act to localize the complex and regulate INa3. With the onset of the action potential (AP) in the myocardium the peak INa rapidly rises and decays to nearly zero over several ms. This INa spike underlies excitability and conduction in working myocardium and the Purkinje conduction system. In contrast to peak INa, late INa is a small inward current, usually less than 0.5% of peak INa, that flows throughout the action potential (AP) plateau. Although the amplitude of late INa is small, it plays a role to maintain the AP plateau because competing repolarizing potassium currents are also small. Increased late INa can directly affect cardiac
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DOI: 10.1161/CIRCULATIONAHA.115.017980
electrophysiology by prolonging refractoriness and predisposing to triggered activity as early after-depolarizations or EADs, observed clinically as long QT arrhythmia. Because late INa flows for much longer time than peak INa (~300 to 400 ms for late INa) it is predicted to play a greater role in Na+ loading than peak INa4. Increased Na+ loading increases intracellular Ca2+ levels through effects on Na+-Ca2+ exchange, and thereby affects contractility and relaxation5. Increased intracellular Ca2+ levels affect the electrophysiology of the cell via a number of mechanisms including delayed after-depolarizations, or DADs. Late INa is increased under many conditions including inherited disorders such as in inherited long QT syndromes (LQT 3, 9, 10, 12), and also in acquired conditions, as in hypertrophy, heart failure, ischemia and diabetes, where it plays roles in the pathogenesis of arrhythmia, heart failure, and angina6 aand nd it it has has attracted much attention as a therapeutic drug target7,8,9. Therefore understanding the properties and pathways path pa thwa th ways wa ys reg gul u ating late INa has the potential al to to help us understandd the th pathogenesis and regulating pprovide rovvide avenues avenuees for fo or treatment treaatm tr tmen entt of many en man a y disease disea eaasee processes pro ocessses in in clinical c in cl nic i al ccardiology. arddiolog ar di og ogy. y y. about In this thi hiss commentary hi co ommen ntary we we consider c ns co nsid i er key key y unanswered unnansswere reed and an nd partially part pa rtiaallly answered rt anssweereed ed questions que uesstions ue nss abo out late ate INa, and and discuss disc di scus sc uss ss how ho th thee genetically gene ge neti ne tica ti call ca lly engineered ll engi en gine gi neer ne ered er ed mi mice ce ddeveloped evel ev elop el oped op ed aand nd ccharacterized hara ha ract ra cter ct eriz er ized ed bby y Hu H Hund und nd and colleagues1 have addressed or could be used to address them. What are the signaling pathways that regulate late INa? How do they interact? Two pathways that enhance late INa act by post-translational modification of Scn5a involve CaMKII dependent phosphorylation10 and nNOS dependent nitrosylation.11 A third pathway that may involve direct phosphorylation of Scn5a or other regulatory protein, involves phosphoinositide 3-kinase (PI3K), which acts to suppress late INa.12 The CaMKII pathway is presently the most studied and best defined with the key phosphorylation site affecting late INa known to be S571. The nNOS pathway appears to involve direct nitrosylation of the Scn5a
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channel, but the Cys site(s) have not yet been determined. Whether and how these different pathways interact are unknown. Are they independent and additive? Do they share common features? It is not known whether or not the PI3K pathway acts directly by phosphorylation of Scn5a12, or whether it may somehow involve the CaMKII or nNOS or other pathways. Although these questions were not directly addressed in the present study, it is interesting to note that the S571A mouse retains a signficant proportion of WT late INa (Fig. 2 in Glynn et al. 1) suggesting a componenet of late INa that is not regulated by the S571 site. The S571 mouse models should be useful to address other questions about pathway interactions. For example, would inhibition of the PI3K pathway result in an increase in late INa in the S571A model? If not, this would support upport the idea that PI3K activation ultimately acts by supressing phosphorylation on of of S571. S571 S5 71.. In 71 addition to the above named pathways PKC-dependent phosphorylation at S1503 altered INa kine eti tics cs in in a way w y that wa th enhances a type of late INaa called cal alled “window current” curren nt”13 and PKC inhibition kinetics bblocked bloc l cked increased increase sed late se laate INa th that at w was as ccaused au useed by ccalcium alciium lloading oadi oa ding di ng tthe he ccell ell el ll 14, suggesting sugg ggeesti gg esti ting ng roles rol oles ol e for for PKC PK KC that m may ay bee iinteract nterracct wit with ithh th it tthee Ca C CaMKII MKII ppathway. MK athw wayy. Th The S5 S571 S571A 1A an and nd S5 S S571E 571E Em models odeels wi will illl be useful us usef sef eful ull ttools ools oo ls tto o fu ffurther urt rthe rt herr de he defi define fine fi ne th thee re rela relationships lati la tion ti onsh on ship sh ipss an ip andd re rela relative lati la tive ti vee iimportance mpor mp orta or tanc ta ncee am nc amon among ongg th on thes these esee pathways. es path pa thwa th ways ays. s What signaling pathways are involved in inherited and acquired diseases with increased late INa? Enhanced late INa occurs in numerous inherited cardiac disorders (mutations in the Scn5a complex for LQT3, LQT9. LQT19, LQT12), and in acquired conditions (hypertrophy, heart failure, ischemia, diabetes), and can also arise due to changes in metabolites and other molecules (acidosis, carbon monoxide (CO), reative oxygen species (ROS) and drugs (PI3K inhibitors)) 9. The detailed mechanisms for the causes of late INa in disease and the pathways involved have been investigated in only a few of these diseases. Activation of the nNOS pathway to increase
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DOI: 10.1161/CIRCULATIONAHA.115.017980
late INa has been implicated in the pathogenesis of LQT9 involving caveolin3 mutations15 and LQT12 involving Į1-syntrophin mutations11. An important finding in the present study1 is that stress induced heart failure in the S571A mouse failed to develop the increased late INa seen in wild-type mice1, strongly supporting the idea that phosphorylation of S571 via the CaMKII pathway is required for the late INa in this model of heart failure. Further experiments in this model could extend these important insights about the role of the CamKII pathway in causes of late INa. For example, would late INa be enhanced by ischemia, CO, ROS, PI3K inhibitors in the S571A mouse? If not, this would provide evidence that they all work through the CaMKII pathway. What are the biophysical and structure function mechanisms for late INa? Overall the detailed biophysical mechanism(s) for late INa at the level of Scn5a are not clear. The prev vai aili ling li ng iidea dea iss tthat de h t it involves the inactivationn ggate ha a e on the D3-4 link at ker, er, orr in the inactivation prevailing linker, receptor eceeptor involving invollvi ving n the ng he S4-5 S44-55 linker liink nker err and and residues reesid id due ues onn S5. S5. But But LQT3 LQT QT33 mutations muta mu taatioons occur occu oc curr over cu overr m much uchh of uc of the he Scn5a Sc to topography. opo p grrap phyy. A lack ck ooff “h “hot hot o sspots” poots”” su suggests ugg ggessts ccomplexity omppleexit exi y in in tthe hee sstructures truc uctu uc ures af aff affecting fecttin ng complete comp co mple mp lete le te iinactivation nact na ctiv ct ivat atio at ionn of INa. In io In particular, part pa rtic rt icul ic ular lar ar, how ho does does phosphorylation pho hosp spho sp hory ho ryla lati la tion ti on of of S571 S571 increase inc ncre reas re asee la as late te INa? Is it linked in some way to inactivation structures? While the present study does not address these structure-function and biophysical issues directly, the finding that late INa can be regulated by phosphorylation at S571 independently of other gating changes is of interest, and must be accounted for by any proposed structure-function model. Two LQT3 mutations close to S571 were postulated to cause late INa by mimicking the charge near this site 16 and provide additional clues to the structure-function of late INa. Other nearby CaMKII-dependent phosphorylation sites (S516 and T594) do affect kinetics of gating but do not appear to contribute specifically to late INa.17 These key and interesting findings might be investigated by additional
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DOI: 10.1161/CIRCULATIONAHA.115.017980
electrophysiological studies including single channel analysis of the S571 knock-in mouse lines. Another key observation was the rate-dependent decrease in late INa in these mice. This property was seen with the canonical LQT3 mutation delta KPQ18 , and has been postulated to be protective. The late INa found in some Scn5a mutations, such as those found in sudden Infant death syndrome (SIDS)19, do not have this property and may account for greater lethality. Is physiological late INa (in contrast to the enhanced late INa found in inherited and acquired disorders) important for regulating excitability and contractility in the absence of disease? Most studies of late INa have been conducted in models of pathologically enhanced late INa, but late ate INa is also present in normal hearts (usually 0.2 to 0.5% of peak INa). Is this physiological phy hysi hy siol si olog ol ogic og ical ic al late late INa important for normal electrophysiology and regulation of contractility? Might drugs that bloc ck to tooo mu much ooff the late INa lead to undesired eeffects? ffeects? The recognitio ff recognition on th tthat at late INa plays a role block inn normal normal cardiac cardia iacc physiology ia phhysio ysiolo olo logy gy goes goe oess back bac ~50 50 years yearss when when en it it was wa shown show sh ownn that ow that the tha he selective sel elec ecti ec t ve N Naa+ 20 channel ch han anne n l bloc blocker occke k r te tetr tetrodotoxin t oddottoxin n sh shortened horten enedd the en th AP pplateau laateeau u inn no normal orm mall m myocytes yocy yo ytes20 . But B t in normal Bu norrmal
abb bbit it and and rat rat hearts hea eart rtss specific rt spec sp ecif ec ific if ic llate atee INa blockers at blo lock cker ck erss had er had no important imp mpor orta or tant ta nt effects effe ef fect fe ctss on contractility ct con ontr trac tr acti ac tili ti lity li ty and and rabbit conduction.21 Small but significant decreases in ejection fraction were observed in the S571A mouse1, supporting the idea that CaMKII-dependent late INa has importance in regulating contractility in normal heart. But “S571A APD was not significantly different than WT at baseline” 1 suggesting modest if any effects on electrophysiology. It is possible that the complex pathways for regulation of late INa evolved to deal exclusively with stress or pathological conditions, but it is more likely that these physiological levels of late INa are under tight regulatory control for important reasons related to normal cardiac physiology. More studies are needed on this less well studied issue.
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Can pathologically enhanced late INa be better selectively targeted based on pathway mechanism? The authors of the present study1 emphasized how CaMKII-dependent S571 phosphorylation specifically regulates late INa and may represent an attractive target for blocking pathological late INa. Currently used drugs such as flecainide, amiodarone, and ranolazine are all pore blockers and would presumably block late INa regardless of mechanism of generating late INa6. They appear to get their selectivity to block late INa over peak INa because of state-dependent block. Even more selective blockers of late INa are in development22. Regulating the phosphorylation S571 could in theory be a novel and important specific regulator of late INa but it is not yet clear iff small molecules can be found for this target. Caveats and importance As tthe he aauthors utho ut hors ho r off th the present study correctly poin point in nt oout, ut, a mouse model m may a not be completely ay translatable ran nslatable to human hum hu man physiology. phys ph hys ysiiolo iolo logy gy.. Despite gy Desspiite this De th s limitation lim mittatio on this this study stu tudy tu dyy1 hhas as ggenerated ener erat er ated at ed insights ins n ig ight htss in ht into o unanswered un nan answ s ered d questions quesstionss aabout boutt the he regu regulatory gulaato gu ory y ppathways atthway ys an andd ch characteristics harac a terrissti ac t cs ooff bo both t pa pathological athholo ogiical an and nd physiological phys ph ysio sio iolo logi lo gica gi call la ca late te INa , and and the the models mode mo dels de ls developed dev evel elop el oped op ed in in these thes th esee studies es stud st udie die iess have havee the the potential pot oten enti en tial ti al to to generate gene ge nera ne rate ra te more insights.
Conflict of Interest Disclosures: None.
References: 1. Glynn P, Musa H, Wu X, Unudurthi S, Little S, Qian L, Wright PJ, Radwanski PB, Gyorke S, Mohler PJ, Hund TJ. Voltage-gated sodium channel phosphorylation at Ser571 regulates late current, arrhythmia, and cardiac function in vivo. Circulation. 2015;132:XX-XXX. 2. Hund TJ, Koval OM, Li J, Wright PJ, Qian L, Snyder JS, Gudmundsson H, Kline CF, Davidson NP, Cardona N, Rasband MN, Anderson ME, Mohler PJ. A beta(IV)-spectrin/CaMKII signaling complex is essential for membrane excitability in mice. J Clin Invest. 2010;120:35083519.
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3. Abriel H. Cardiac sodium channel Na(v)1.5 and interacting proteins: Physiology and pathophysiology. J Mol Cell Cardiol. 2010;48:2-11. 4. Makielski JC, Farley AL. Na(+) current in human ventricle: implications for sodium loading and homeostasis. J Cardiovasc Electrophysiol. 2006; 17 Suppl 1:S15-S20. 5. Bers DM, Chen-Izu Y. Sodium and calcium regulation in cardiac myocytes: from molecules to heart failure and arrhythmia. J Physiol. 2015;593:1327-1329. 6. Makielski JC. Late sodium current: A mechanism for angina, heart failure, and arrhythmia. Trends Cardiovasc Med. 2015 May 22. pii: S1050-1738(15)00151-6. doi: 10.1016/j.tcm.2015.05.006. [Epub ahead of print]. 7. Antzelevitch C, Nesterenko V, Shryock JC, Rajamani S, Song Y, Belardinelli L. The role of late I Na in development of cardiac arrhythmias. Handb Exp Pharmacol. 2014;221:137-168. 8. Maltsev VA, Undrovinas A. Late sodium current in failing heart: friend or foe? Prog Biophys 2008;96:421-451. Mol Biol. 2008;96:421 451. Na(+) 9. Belardinelli L, Giles WR, Rajamani S, Karagueuzian HS, Shryock JC. Cardiac llate atee Na at Na(+ (+)) (+ current: proarrhythmic effects, roles in long QT syndromes, and pathological relationship to CaMKII and oxidative stress. Heart Rhythm. 2015;12:440-448. 10. S,, Dy Dybkova 10 0. Wagner Wagner S ybkova N, Rasenack EC, Jacobshagen Jacobsha hagen C, Fabritz ha tz L, Kirchhof P, Maier SK, Zhang Hasenfuss G,, Br Brown Bers DM, Maier LS. Ca2+/calmodulin-dependent protein Z haang T, Hasen nfusss G Brow ownn JH ow JH, Be Ber rs DM M, M aiierr LS S. Ca Ca2+ 2+/ccal 2+ a mo modu dulinn-deepe du pend nden nd ent pr en prot tei einn regulates cardiac channels. Invest. 2006;116:3127-3138. kkinase ki naase II regulate es car rdiiac Na+ Na+ channe elss. J Cl Clin i Inv nvvest. 20 200 06;;116 6:3 3127--31388. 11. Ueda Ueda K, K, Valdivia Vaaldiv ldivia via C, C, Medeiros-Domingo Medeeir iros os-Dom os min ingo go A, A, Tester Test Te ste ter DJ, DJ, Vatta Vatta at M, M, Farrugia Farr Fa rrug ugia G, ug G, Ackerman Ackeerman Ac rman n MJ, M J, Makielski through activation Maki Ma kiel ki elsk el skii JC. sk JC Syntrophin Synt Sy ntro nt roph ro phin ph in mutation mutat uttat atiion associated ass ssoc ocia oc iate ia tedd with te wiith llong ongg QT ssyndrome on ynndr yndr drom omee th om thro roug ro ugh gh ac acti tiva ti vati ati tion on ooff th thee nNOS-SCN5A macromolecular complex. Proc Natl Acad Sci U S A. 2008; 105:9355-9360. 12. Ballou LM, Lin RZ, Cohen IS. Control of cardiac repolarization by phosphoinositide 3kinase signaling to ion channels. Circ Res. 2015;116:127-137. 13. Qu Y, Rogers JC, Tanada TN, Catterall WA, Scheuer T. Phosphorylation of S1505 in the cardiac Na+ channel inactivation gate is required for modulation by protein kinase C. J Gen Physiol. 1996;108:375-379. 14. Wu Y, Wang L, Ma J, Song Y, Zhang P, Luo A, Fu C, Cao Z, Wang X, Shryock JC, Belardinelli L. Protein kinase C and Ca(2+) -calmodulin-dependent protein kinase II mediate the enlarged reverse INCX induced by ouabain-increased late sodium current in rabbit ventricular myocytes. Exp Physiol. 2015;100:399-409. 15. Cheng J, Valdivia CR, Vaidyanathan R, Balijepalli RC, Ackerman MJ, Makielski JC. Caveolin-3 suppresses late sodium current by inhibiting nNOS-dependent S-nitrosylation of SCN5A. J Mol Cell Cardiol. 2013;61:102-110.
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16. Koval OM, Snyder JS, Wolf RM, Pavlovicz RE, Glynn P, Curran J, Leymaster ND, Dun W, Wright PJ, Cardona N, Qian L, Mitchell CC, Boyden PA, Binkley PF, Li C, Anderson ME, Mohler PJ, Hund TJ. Ca2+/Calmodulin-Dependent Protein Kinase II-Based Regulation of Voltage-Gated Na+ Channel in Cardiac Disease. Circulation. 2012;126:2084-2094. 17. Ashpole NM, Herren AW, Ginsburg KS, Brogan JD, Johnson DE, Cummins TR, Bers DM, Hudmon A. Ca2+/calmodulin-dependent protein kinase II (CaMKII) regulates cardiac sodium channel NaV1.5 gating by multiple phosphorylation sites. J Biol Chem. 2012;287:19856-19869. 18. Nagatomo T, January CT, Ye B, Abe H, Nakashima Y, Makielski JC. Rate-dependent QT shortening mechanism for the LQT3 DeltaKPQ mutant. Cardiovasc Res. 2002;54:624-629. 19. Ackerman MJ, Siu BL, Sturner WQ, Tester DJ, Valdivia CR, Makielski JC, Towbin JA. Postmortem molecular analysis of SCN5A defects in sudden infant death syndrome. JAMA. 2001;286:2264-2269. 20. Dudel J, Peper K, Rudel R, Trautwein W. Effect of tetrodotoxin on membrane currents in 1967;213:296-297. mammalian cardiac fibres. Nature. 1967;213:296 297. Selective 21. Fernandes S, Hoyer K, Liu G, Wang WQ, Dhallaa AK, Belardinelli L, Rajamani Rajaman an ni S. S elec el ecti ec tive ti ve inhibition nhibition of the late sodium current has no adverse effect on electrophysiological or contractile function of the normal heart. J Cardiovasc Pharmacol. 2014;63:512-519. 22. Belardinelli 22 2. B elardin nelli L, Liu G, Smith-Maxwell C, Wang Wanng WQ, El-Bizri El-Bizrri N, N, Hirakawa R, Karpinski S, Lii CH, Zablocki Yao L,, D Dhalla Rajamani S,, L CH, Hu L, Li Li XJ, XJ, Crumb Crum Crum umbb W, Wu L, L, Koltun Kolt ltun lt un D, Za abloc ocki oc ki JJ,, Ya Y oL halla AK ha AK, Ra Raja j mani ja ni S Shryock potent, selective inhibitor late sodium suppresses S Sh ryock JC. A no ry nnovel, vel, po otentt, aand nd selec ctiive inh nhibittorr off ccardiac nh arddiaac la ar atee sodi ium m ccurrent urrentt su upppreessess experimental arrhythmias. Pharmacol Exp Ther. ex xpe peri r mental al arr rhy hythm miaas. J Ph Phar a ma maco ol Ex E pT herr. 22013;344:23-32. 0113;;344 ;344 4:2 233 32 32.
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Late INa in the Heart: Physiology, Pathology, and Pathways Jonathan C. Makielski and John W. Kyle Circulation. published online July 17, 2015; Circulation is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright © 2015 American Heart Association, Inc. All rights reserved. Print ISSN: 0009-7322. Online ISSN: 1524-4539
The online version of this article, along with updated information and services, is located on the World Wide Web at: http://circ.ahajournals.org/content/early/2015/07/17/CIRCULATIONAHA.115.017980
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Late INa in the Heart: Physiology, Pathology, and Pathways
Running title: Makielski et al.; Late INa regulatory pathways Jonathan C. Makielski, MD; John W. Kyle, PhD
Dept of Medicine, Division of Cardiovascular Medicine, University of Wiscon Wisconsinnsi sinn- M Madison, adis ad ison is on, on Madison, WI
Address A dd dress for Correspondence: Corrres Co rr spond pond nden nce ce:: Jonathan Jo Jona o athan C. Makielski, M kieelskii, MD Ma Depa part pa rtme rt ment me nt of Medicine, Meddici Me cine ne, e Division Divi visi vi sion si on of Cardiovascular Card Ca rdio rd iova io vasc va scul sc ular ar M edic ed icin ic inee in Department Medicine University of Wisconsin- Madison 600 Highland Ave H4/5 Madison, WI 53792 Tel: 608-262-2075 Fax: 608-263-0405 E-mail: [email protected]
Journal Subject Codes: Atherosclerosis:[145] Genetically altered mice, Atherosclerosis:[138] Cell signaling/signal transduction, Basic science research:[132] Arrhythmias - basic studies, Atherosclerosis:[148] Heart failure - basic studies, Myocardial biology:[104] Structure
Key words: Editorial, long QT syndrome, calcium/calmodulin-dependent protein kinase II, arrhythmia, sodium current, Scn5a
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DOI: 10.1161/CIRCULATIONAHA.115.017980
In this issue of Circulation, Hund and colleagues1 make an important contribution to understanding the physiological and pathophysiological roles of late sodium current (INa) in the heart, with a focus on a key pathway regulating late INa amplitude. They conducted well designed and detailed studies with two new genetically engineered mouse lines: one a S571A mouse that ablates phosphorylation by Ca2+/calmodulin-dependent kinase II (CaMKII)2 selectively at serine 571 in the cardiac Na channel pore-forming protein Scn5a, and the other a S571E mouse that mimics phosphorylation at serine 571. Serine 571 was shown previously to be a target for phosphorylation by CaMKII and this phosphorylation enhanced late INa2. The present studies in “knock-in” mice expressing either S571A or S571E have distinct advantages over other earlier studies tudies in heterologous expression systems including cultured myocyte models because bec ecau ec ause au se they the hey y allow the study of whole animal and organ phenotypes, as well as the study of cellular and molecular mole leecu cula larr bi la biop biophysical o hyysi s cal properties in a more nativ native ve en environment. nvironment. What th these hese new in vivo studies reveal eveeal is that des despite espite i e tthe he ex exte extensive tens te nsiive ns ive nnetwork etw twoorkk of CaMKII tw CaM MKIII targets, targ ta a getts, phosphorylation phoosp spho horyylati ho tion ti on ooff S S571 571 selectively regulates ele l ct ctively re reg gula late a es late laatee INa aand nd inn pa pparticular rticculaar eenhanced nhan an ncedd llate atee INa inn failing fail ilin il ng hea heart. art. t. Pe ak INa iiss th thee large larg la rgee inward rg inwa in ward ard current curre urrre rent nt fflowing lowi lo wing ing m mainly ainl ai nly th nl thro through roug ro ugh gh th thee ca card cardiac rdia rd iacc Na+ cchannel ia hann ha nnel nn el pore por oree Peak formed by Scn5a, which is part of a larger sodium channel macromolecular complex. Members of this macromolecular complex act to localize the complex and regulate INa3. With the onset of the action potential (AP) in the myocardium the peak INa rapidly rises and decays to nearly zero over several ms. This INa spike underlies excitability and conduction in working myocardium and the Purkinje conduction system. In contrast to peak INa, late INa is a small inward current, usually less than 0.5% of peak INa, that flows throughout the action potential (AP) plateau. Although the amplitude of late INa is small, it plays a role to maintain the AP plateau because competing repolarizing potassium currents are also small. Increased late INa can directly affect cardiac
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DOI: 10.1161/CIRCULATIONAHA.115.017980
electrophysiology by prolonging refractoriness and predisposing to triggered activity as early after-depolarizations or EADs, observed clinically as long QT arrhythmia. Because late INa flows for much longer time than peak INa (~300 to 400 ms for late INa) it is predicted to play a greater role in Na+ loading than peak INa4. Increased Na+ loading increases intracellular Ca2+ levels through effects on Na+-Ca2+ exchange, and thereby affects contractility and relaxation5. Increased intracellular Ca2+ levels affect the electrophysiology of the cell via a number of mechanisms including delayed after-depolarizations, or DADs. Late INa is increased under many conditions including inherited disorders such as in inherited long QT syndromes (LQT 3, 9, 10, 12), and also in acquired conditions, as in hypertrophy, heart failure, ischemia and diabetes, where it plays roles in the pathogenesis of arrhythmia, heart failure, and angina6 aand nd it it has has attracted much attention as a therapeutic drug target7,8,9. Therefore understanding the properties and pathways path pa thwa th ways wa ys reg gul u ating late INa has the potential al to to help us understandd the th pathogenesis and regulating pprovide rovvide avenues avenuees for fo or treatment treaatm tr tmen entt of many en man a y disease disea eaasee processes pro ocessses in in clinical c in cl nic i al ccardiology. arddiolog ar di og ogy. y y. about In this thi hiss commentary hi co ommen ntary we we consider c ns co nsid i er key key y unanswered unnansswere reed and an nd partially part pa rtiaallly answered rt anssweereed ed questions que uesstions ue nss abo out late ate INa, and and discuss disc di scus sc uss ss how ho th thee genetically gene ge neti ne tica ti call ca lly engineered ll engi en gine gi neer ne ered er ed mi mice ce ddeveloped evel ev elop el oped op ed aand nd ccharacterized hara ha ract ra cter ct eriz er ized ed bby y Hu H Hund und nd and colleagues1 have addressed or could be used to address them. What are the signaling pathways that regulate late INa? How do they interact? Two pathways that enhance late INa act by post-translational modification of Scn5a involve CaMKII dependent phosphorylation10 and nNOS dependent nitrosylation.11 A third pathway that may involve direct phosphorylation of Scn5a or other regulatory protein, involves phosphoinositide 3-kinase (PI3K), which acts to suppress late INa.12 The CaMKII pathway is presently the most studied and best defined with the key phosphorylation site affecting late INa known to be S571. The nNOS pathway appears to involve direct nitrosylation of the Scn5a
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channel, but the Cys site(s) have not yet been determined. Whether and how these different pathways interact are unknown. Are they independent and additive? Do they share common features? It is not known whether or not the PI3K pathway acts directly by phosphorylation of Scn5a12, or whether it may somehow involve the CaMKII or nNOS or other pathways. Although these questions were not directly addressed in the present study, it is interesting to note that the S571A mouse retains a signficant proportion of WT late INa (Fig. 2 in Glynn et al. 1) suggesting a componenet of late INa that is not regulated by the S571 site. The S571 mouse models should be useful to address other questions about pathway interactions. For example, would inhibition of the PI3K pathway result in an increase in late INa in the S571A model? If not, this would support upport the idea that PI3K activation ultimately acts by supressing phosphorylation on of of S571. S571 S5 71.. In 71 addition to the above named pathways PKC-dependent phosphorylation at S1503 altered INa kine eti tics cs in in a way w y that wa th enhances a type of late INaa called cal alled “window current” curren nt”13 and PKC inhibition kinetics bblocked bloc l cked increased increase sed late se laate INa th that at w was as ccaused au useed by ccalcium alciium lloading oadi oa ding di ng tthe he ccell ell el ll 14, suggesting sugg ggeesti gg esti ting ng roles rol oles ol e for for PKC PK KC that m may ay bee iinteract nterracct wit with ithh th it tthee Ca C CaMKII MKII ppathway. MK athw wayy. Th The S5 S571 S571A 1A an and nd S5 S S571E 571E Em models odeels wi will illl be useful us usef sef eful ull ttools ools oo ls tto o fu ffurther urt rthe rt herr de he defi define fine fi ne th thee re rela relationships lati la tion ti onsh on ship sh ipss an ip andd re rela relative lati la tive ti vee iimportance mpor mp orta or tanc ta ncee am nc amon among ongg th on thes these esee pathways. es path pa thwa th ways ays. s What signaling pathways are involved in inherited and acquired diseases with increased late INa? Enhanced late INa occurs in numerous inherited cardiac disorders (mutations in the Scn5a complex for LQT3, LQT9. LQT19, LQT12), and in acquired conditions (hypertrophy, heart failure, ischemia, diabetes), and can also arise due to changes in metabolites and other molecules (acidosis, carbon monoxide (CO), reative oxygen species (ROS) and drugs (PI3K inhibitors)) 9. The detailed mechanisms for the causes of late INa in disease and the pathways involved have been investigated in only a few of these diseases. Activation of the nNOS pathway to increase
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DOI: 10.1161/CIRCULATIONAHA.115.017980
late INa has been implicated in the pathogenesis of LQT9 involving caveolin3 mutations15 and LQT12 involving Į1-syntrophin mutations11. An important finding in the present study1 is that stress induced heart failure in the S571A mouse failed to develop the increased late INa seen in wild-type mice1, strongly supporting the idea that phosphorylation of S571 via the CaMKII pathway is required for the late INa in this model of heart failure. Further experiments in this model could extend these important insights about the role of the CamKII pathway in causes of late INa. For example, would late INa be enhanced by ischemia, CO, ROS, PI3K inhibitors in the S571A mouse? If not, this would provide evidence that they all work through the CaMKII pathway. What are the biophysical and structure function mechanisms for late INa? Overall the detailed biophysical mechanism(s) for late INa at the level of Scn5a are not clear. The prev vai aili ling li ng iidea dea iss tthat de h t it involves the inactivationn ggate ha a e on the D3-4 link at ker, er, orr in the inactivation prevailing linker, receptor eceeptor involving invollvi ving n the ng he S4-5 S44-55 linker liink nker err and and residues reesid id due ues onn S5. S5. But But LQT3 LQT QT33 mutations muta mu taatioons occur occu oc curr over cu overr m much uchh of uc of the he Scn5a Sc to topography. opo p grrap phyy. A lack ck ooff “h “hot hot o sspots” poots”” su suggests ugg ggessts ccomplexity omppleexit exi y in in tthe hee sstructures truc uctu uc ures af aff affecting fecttin ng complete comp co mple mp lete le te iinactivation nact na ctiv ct ivat atio at ionn of INa. In io In particular, part pa rtic rt icul ic ular lar ar, how ho does does phosphorylation pho hosp spho sp hory ho ryla lati la tion ti on of of S571 S571 increase inc ncre reas re asee la as late te INa? Is it linked in some way to inactivation structures? While the present study does not address these structure-function and biophysical issues directly, the finding that late INa can be regulated by phosphorylation at S571 independently of other gating changes is of interest, and must be accounted for by any proposed structure-function model. Two LQT3 mutations close to S571 were postulated to cause late INa by mimicking the charge near this site 16 and provide additional clues to the structure-function of late INa. Other nearby CaMKII-dependent phosphorylation sites (S516 and T594) do affect kinetics of gating but do not appear to contribute specifically to late INa.17 These key and interesting findings might be investigated by additional
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electrophysiological studies including single channel analysis of the S571 knock-in mouse lines. Another key observation was the rate-dependent decrease in late INa in these mice. This property was seen with the canonical LQT3 mutation delta KPQ18 , and has been postulated to be protective. The late INa found in some Scn5a mutations, such as those found in sudden Infant death syndrome (SIDS)19, do not have this property and may account for greater lethality. Is physiological late INa (in contrast to the enhanced late INa found in inherited and acquired disorders) important for regulating excitability and contractility in the absence of disease? Most studies of late INa have been conducted in models of pathologically enhanced late INa, but late ate INa is also present in normal hearts (usually 0.2 to 0.5% of peak INa). Is this physiological phy hysi hy siol si olog ol ogic og ical ic al late late INa important for normal electrophysiology and regulation of contractility? Might drugs that bloc ck to tooo mu much ooff the late INa lead to undesired eeffects? ffeects? The recognitio ff recognition on th tthat at late INa plays a role block inn normal normal cardiac cardia iacc physiology ia phhysio ysiolo olo logy gy goes goe oess back bac ~50 50 years yearss when when en it it was wa shown show sh ownn that ow that the tha he selective sel elec ecti ec t ve N Naa+ 20 channel ch han anne n l bloc blocker occke k r te tetr tetrodotoxin t oddottoxin n sh shortened horten enedd the en th AP pplateau laateeau u inn no normal orm mall m myocytes yocy yo ytes20 . But B t in normal Bu norrmal
abb bbit it and and rat rat hearts hea eart rtss specific rt spec sp ecif ec ific if ic llate atee INa blockers at blo lock cker ck erss had er had no important imp mpor orta or tant ta nt effects effe ef fect fe ctss on contractility ct con ontr trac tr acti ac tili ti lity li ty and and rabbit conduction.21 Small but significant decreases in ejection fraction were observed in the S571A mouse1, supporting the idea that CaMKII-dependent late INa has importance in regulating contractility in normal heart. But “S571A APD was not significantly different than WT at baseline” 1 suggesting modest if any effects on electrophysiology. It is possible that the complex pathways for regulation of late INa evolved to deal exclusively with stress or pathological conditions, but it is more likely that these physiological levels of late INa are under tight regulatory control for important reasons related to normal cardiac physiology. More studies are needed on this less well studied issue.
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Can pathologically enhanced late INa be better selectively targeted based on pathway mechanism? The authors of the present study1 emphasized how CaMKII-dependent S571 phosphorylation specifically regulates late INa and may represent an attractive target for blocking pathological late INa. Currently used drugs such as flecainide, amiodarone, and ranolazine are all pore blockers and would presumably block late INa regardless of mechanism of generating late INa6. They appear to get their selectivity to block late INa over peak INa because of state-dependent block. Even more selective blockers of late INa are in development22. Regulating the phosphorylation S571 could in theory be a novel and important specific regulator of late INa but it is not yet clear iff small molecules can be found for this target. Caveats and importance As tthe he aauthors utho ut hors ho r off th the present study correctly poin point in nt oout, ut, a mouse model m may a not be completely ay translatable ran nslatable to human hum hu man physiology. phys ph hys ysiiolo iolo logy gy.. Despite gy Desspiite this De th s limitation lim mittatio on this this study stu tudy tu dyy1 hhas as ggenerated ener erat er ated at ed insights ins n ig ight htss in ht into o unanswered un nan answ s ered d questions quesstionss aabout boutt the he regu regulatory gulaato gu ory y ppathways atthway ys an andd ch characteristics harac a terrissti ac t cs ooff bo both t pa pathological athholo ogiical an and nd physiological phys ph ysio sio iolo logi lo gica gi call la ca late te INa , and and the the models mode mo dels de ls developed dev evel elop el oped op ed in in these thes th esee studies es stud st udie die iess have havee the the potential pot oten enti en tial ti al to to generate gene ge nera ne rate ra te more insights.
Conflict of Interest Disclosures: None.
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Late INa in the Heart: Physiology, Pathology, and Pathways Jonathan C. Makielski and John W. Kyle Circulation. published online July 17, 2015; Circulation is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright © 2015 American Heart Association, Inc. All rights reserved. Print ISSN: 0009-7322. Online ISSN: 1524-4539
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