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Is phosphorylation key to the fight or flight response and heart failure? Thomas Eschenhagen

Department of Experimental Pharmacology and Toxicology, Cardiovascular Research Center Hamburg, University Medical Center Hamburg Eppendorf, Hamburg, Germany.

In situations of stress the heart beats faster and stronger. According to changes in excitation-contraction coupling, Marks and colleagues, this response is, to a large extent, the consequence of i.e., the relationship between the cardiac facilitated Ca2+ release from intracellular Ca2+ stores via ryanodine receptor 2 action potential and myofilament activa- (RyR2), thought to be due to catecholamine-induced increases in RyR2 tion (Figure 1 of this commentary). When phosphorylation at serine 2808 (S2808). If catecholamine stimulation is the cell depolarizes during a cardiac action sustained (for example, as occurs in heart failure), RyR2 becomes hyper- potential, L-type Ca2+ channels (LTCCs) phosphorylated and “leaky,” leading to arrhythmias and other pathology. open, allowing Ca2+ to enter the cell. This This “leaky RyR2 hypothesis” is highly controversial. In this issue of the so-called trigger Ca2+ induces a much larger JCI, Marks and colleagues report on two new mouse lines with mutations Ca2+ release from intracellular Ca2+ stores, in S2808 that provide strong evidence supporting their theory. Moreover, known as the (SR), the experiments revealed an influence of redox modifications of RyR2 that via large tetrameric ryanodine-sensitive may account for some discrepancies in the field. channels, referred to as ryanodine receptor 2 (RyR2). The increase in Ca2+ concentra- The heart has a remarkable capacity to react node cells (the cells that generate the action tion initiates a conformational change in to altered demand by changing the rate at potentials that trigger cardiac contraction) the myofilaments and thereby contrac- which it beats and the force with which it and thus acceleration of heart rate (i.e., it tion. Removal of Ca2+ from the cytosol via contracts, thereby changing its output. has a “positive chronotropic effect”) and the sarcoplasmic/endoplasmic reticulum Both the reduction of cardiac output in stronger contraction (i.e., it has a “posi- Ca2+ ATPase (SERCA) and the Na+/Ca2+ phases of rest and its increase in physical tive inotropic effect”) and faster relaxation exchanger (NCX) in the plasmalemma and emotional exercise (the fight or flight (i.e., it has a “positive lusitropic effect”) in reverses the process. Importantly, the response) are essential for normal body working myocytes. In chronic heart failure, amount of Ca2+ entering the cell is, under homeostasis and long-term survival. It is one of the most frequent life-threatening normal conditions, exactly matched by not surprising therefore that cardiac rate diseases in Western societies, norepineph- the amount of Ca2+ leaving it via the NCX. and force are regulated at multiple levels, rine levels are chronically elevated, which b1-Adrenergic receptors stimulate the sys- extrinsic and intrinsic to the heart, and leads to desensitization of the b-adrenergic tem at numerous levels via PKA (Figure 1 in a highly complex and secured fashion. signalling cascade and blunted responses. of this commentary). Phosphorylation of Stimulation of b1-adrenergic receptors by b-Blockers, introduced by Waagstein and LTCCs increases their open probability, the sympathetic neurotransmitter norepi- colleagues in the mid 1970s, protect the allowing more Ca2+ to enter the cell. Phos- nephrine induces increased production of heart from chronic sympathetic stimula- phorylation of phospholamban (PLB), a the second messenger cAMP. cAMP direct- tion and provide the largest prognostic ben- small that when unphosphorylated ly and indirectly (via activation of PKA) efit for patients with chronic heart failure. inhibits SERCA, leads to disinhibition, i.e., induces faster depolarization in sinoatrial increased reuptake of Ca2+ into the SR. This Cardiac excitation-contraction has at least two consequences: first, more coupling Ca2+ in the SR and therefore more Ca2+ Conflict of interest: The author has declared that no conflict of interest exists. The positive inotropic and lusitropic con- release during , which has a positive 2+ Citation for this article: J Clin Invest. 2010; sequences of b1-adrenergic receptor stimu- inotropic effect; and second, faster Ca 120(12):4197–4203. doi:10.1172/JCI45251. lation in cardiomyocytes are explained by removal from the cytoplasm and thus faster

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Figure 1 The classical view of cardiomyocyte excitation-contraction coupling and its regulation by b-adrenergic receptors. Under unstimulated conditions (black arrows), depolarization during an action potential opens LTCCs in T tubules, allowing Ca2+ to enter the cell. This trig- ger Ca2+ induces a larger Ca2+ release from the SR via RyR2. The increase in Ca2+ concentration initiates conformational changes of the myofilaments and thereby contraction. Removal of Ca2+ via SERCA and NCX reverses the process. Catecholamines stimulate excitation- contraction coupling (red symbols and lettering) by phosphorylating LTCCs (increased Ca2+ influx), PLB (increased Ca2+ reuptake into the SR), and myofilament-based I and -binding protein C (increased relaxation). AC, adenylyl cyclase; Gs, stimulatory G pro- tein. Adapted with permission from Nature (35).

relaxation (i.e., a positive lusitropic effect). also known as calstabin), a small regula- lation affects FKPB12.6 binding. Third, it PKA also phosphorylates the myofilament tory protein that had been known since the seems unlikely and to contradict experi- troponin inhibitor and myosin- early 1990s as a target of two immunosup- mental results (4) that an isolated increase binding protein C, which desensitizes the pressant drugs, tacrolimus (also known as in RyR2 open probability has more than a myofilaments to Ca2+ and facilitates relax- FK506) and rapamycin. Moreover, Marks transient consequence on Ca2+ handling, ation (i.e., positive lusitropic effects). and colleagues provided evidence that because an isolated increase in Ca2+ release RyR2 is chronically hyperphosphorylated from the RyR2 will automatically lead to RyR2 phosphorylation determines in human heart failure and therefore reduced Ca2+ load in the SR and therefore excitation-contraction coupling gain “leaky” (2). Leaky RyR2 channels in turn fast normalization of Ca2+ transients (auto- In the signalling scheme outlined in Fig- could explain both the reduced Ca2+ load of regulation). Thus, leaky RyR2 channels ure 1 of this commentary, which prevailed the SR (which leads to reduced contractile alone, in contrast to super active SERCA (as until the end of the last century, the two force of the failing heart) and the increased in the case of Plb knockout; ref. 1), should major determinants of intracellular Ca2+ risk of spontaneous diastolic Ca2+ release, not affect the basal force of contraction. Of transients and thereby the contractile force which, secondary to Ca2+ extrusion via the course, theoretical arguments can be dis- of the heart were (a) the size of the Ca2+ electrogenic NCX, could explain arrhyth- puted and require experimental validation. current entering via the LTCC (well exem- mias. In this theory, PKA phosphorylation It is now well established that point muta- plified by the negative inotropic effects of RyR2 and the subsequent dissociation tions in RyR2 can cause catecholaminer- of LTCC blockers) and (b) the activity of of FKBP12.6 is the unifying mechanism gic polymorphic ventricular tachycardia SERCA and thus the Ca2+ load of the SR. underlying both the normal fight or flight (CPVT) — an electrophysiological disorder The critical role of the latter was convinc- response and heart failure. of the heart that can cause sudden death ingly demonstrated by the fact that Plb–/– in young people as a result of arrhythmia mice, which have maximal SERCA activ- The controversy about the “leaky — and hence, single-site phosphorylation ity, exhibit higher basal force and reduced RyR2” hypothesis could do so as well. In addition, the auto- inotropic response to isoprenaline (1). In Appealing as Marks’ theory is, the con- regulation argument does not necessarily this scheme, RyR2 was just a passive gate- cept has been challenged and remains hold if one accepts the idea that PKA phos- keeper, opening in response to the trig- controversial (Tables 1 and 2). On the one phorylation of RyR2 is always accompanied ger Ca2+ and releasing Ca2+ at a quantity hand, some theoretical considerations by phosphorylation of LTCC and PLB and proportional to SR Ca2+ concentrations. argue against it. For example, it seems that RyR2 is just one out of (at least) three It is the merit of Marks and colleagues to counterintuitive that phosphorylation at players in the concert. have challenged that view with a whole a single residue in a protein of more than More concerning than theoretical consid- series of papers (starting with their semi- 5,000 amino acids could profoundly affect erations are numerous reports that failed nal paper in 2000; ref. 2), suggesting that channel open probability. Second, S2808, to reproduce important aspects of the PKA also increases the open probability of the proposed site of phosphorylation by data that support the leaky RyR2 hypoth- RyR2 by phosphorylation of serine 2808 PKA, is located in an area distant from the esis and the critical importance of S2808 (S2808) and the subsequent dissociation FKBP12.6/RyR2 interaction site (3), mak- (Tables 1 and 2). (a) Phosphorylation of of FK506-binding protein 12.6 (FKBP12.6; ing it somewhat unlikely that phosphory- RyR2 at S2808 has been found by others to

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Table 1 Overview of the controversy about the role of RyR2 phosphorylation in cardiac function under normal and disease conditions: hyperphosphorylation of RyR2 in heart failure and effect of b-adrenergic stimulation on FKBP12.6 binding and RyR2 open probability

Result/interpretation Method Species Group Year Ref. RyR2 is hyperphosphorylated in heart failure S2808-P increased in HF (approximately IP and back phosphorylation Human Marks 2000 2 increased 800%) due to less PP1 in RyR2 complex No change in S2808-P Back phosphorylation, Dog, human Valdivia 2002 6 phospho-specific Ab No change in S2808-P, S2030 is major PKA site Phospho-specific Ab, Dog Chen 2005 8 phospho-peptide mapping PP1 was up and FKBP12.6 was down in HF, Homogenates and co-IP Rabbit Pogwizd 2005 5 but there was less PP1 and FKBP12.6 in co-IP and Bers with RyR2, more CaMKII in co-IP with RyR2, and S2815-P increased 65%–105% and S2808-P increased 30%–63% in HF S2808-P increased in LV (increased 58%) Post-MI HF Rat ter Keurs 2006 7 but not RV, S2808-P alone had no effect on Ca2+ leak β-Adrenergic stimulation increases RyR2 opening via PKA-phosphorylation at S2808 and dissociation of FKBP12.6 PKA phosphorylation (and hyperphosphorylation Co-IP with or without PKA Dog, human Marks 2000 2 in HF) reduces FKBP12.6 binding and and from NF vs. HF hearts increases Po S2809A mutant binds FKBP12.6 and has Recombinant expression Rabbit Meissner 2003 12 identical properties as WT phospho-mutants Dephosphorylation by PP1 or PP2a increases Permeabilized myocytes, Rat, dog Gyorke 2003 11 Ca2+ leak lipid bilayer Phospho-mutations at 2808 or PKA phosphorylation Recombinant mouse, Mouse, dog Chen 2004 13 does not affect FKBP12.6 binding purified dog RyR2 75% basal S2808 phosphorylation, PKA and SR vesicles, lipid bilayer Sheep Sitsapesan 2006 9 dephosphorylation both increase Po Catalytic subunits of CaMKII and PKA both Permeabilized WT and Plb–/– Mouse Bers 2006 18 increase RyR2 Ca2+ leak, but PKA does so mouse myocytes with and only in WT, indicating exclusive effect on PLB without inhibitors S2808A mice display almost normal function S2808A mice Mouse Valdivia 2007 28 and response to Iso and are not protected after TAC S2808 and S2030 are not located close to 3D reconstruction of mouse RyR2 Mouse Chen 2008 3 FKBP12.6 binding site Normal Iso response in vivo and S2808A mice (Valdivia) Mouse Houser 2008 29 Langendorff-perfused hearts PKA and JTV 519 both reduce FKBP12.6 binding Binding on purified RyR1/2 Rabbit, pig Lai 2010 14 The ratio of FKBP12/12.6 = >10, most FKBP12.6 Permeabilized CM, Rat, mouse Bers 2010 10 was bound, binding unaffected by PKA but GFP-labeled FKBP12/12.6 sensitive to rapamycin

CM, cardiomyocytes; HF, heart failure; JTV 519, experimental drug assumed to stabilize FKBP12.6 binding to RyR2; NF, nonfailing; Iso, isoprenaline; MI, myocardial infarction; PKA-P, phosphorylated PKA; PP1/PP2a, protein phosphatase 1/2a; Po, channel open probability; S2808-P, phosphorylated S2808; TAC, transaortic constriction inducing increased afterload.

be either not altered in heart failure at all unlikely (9). (b) Whereas general consen- PKA had no effect on Ca2+ release in per- or to be only moderately increased (5–8). sus exists that b-adrenergic stimulation meabilized Plb–/– mouse myocytes, i.e., cells Others have reported that 75% of the avail- increases spontaneous Ca2+ release (the in which the SR is maximally loaded with able RyR2 S2808 sites are phosphorylated “Ca2+ leak”) from the SR, the role of RyR2 Ca2+ and one would have expected a par- under normal conditions, making a 9-fold phosphorylation and FKBP12.6 dissocia- ticularly strong effect of increasing RyR2 change in chronic heart failure somewhat tion remains controversial. Importantly, open probability (10). These data suggest

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Table 2 Overview of the controversy about the role of RyR2 phosphorylation in cardiac function under normal and disease conditions: FKBP12.6 binding in CPVT, effect of FKBP12.6 on RyR2 open probability, and the effect of oxidation and nitrosylation on RyR2 open probability and FKBP12.6 binding

Result/interpretation Method Species Group Year Ref. Arrhythmogenic RyR2 mutations go along with reduced FKBP12.6 binding Fkbp12.6–/– mice show exercise-induced Fkbp12.6–/– mice, recombinant Mouse, human Marks 2003 16 arrhythmia, mutated RyR2 shows decreased RyR2 with human mutations FKBP12.6 binding and increased Po in lipid bilayer Mutated RyR2 exhibits normal or slightly Recombinant WT and mutated RyR2, Human Lai 2009 17 increased binding affinity for FKBP12.6, FKBP12.6 binding assay redox state affects binding FKBP12.6 inhibits Ca release from RyR2 Rapamycin decreases FKBP12 binding and Co-IP and lipid bilayer Dog Marks 1996 20 increases RyR2 activity Removal and rebinding of FKBP12.6 to RyR2 SR vesicles, lipid bilayer Dog Fleischer 1996 15 without effect on activity FKBP12.6 overexpression increases SR Ca2+ Adenovirus overexpression Rabbit Smith and 2004 22 load and reduces Ca2+ sparks Hasenfuss Removal of FKBP12.6 has no effect on Coexpression HEK, lipid bilayer, Mouse Chen 2007 24 ryanodine binding, Ca2+ release, or Fkbp12.6–/– mice arrhythmia, Fkbp12.6–/– mice show no stress-induced arrhythmia Transgenic FKBP12.6 overexpression Conditional TG Mouse Mercadier 2008 21 reduces arrhythmia Spark frequency was higher in Fkpb12.6–/– Fkpb12.6–/– Mouse Ji and Kotlikoff 2009 19 mice but they had normal Iso response FKBP12.6 but not FKBP12 slightly inhibits GFP-labeled FKBP12/12.6 Rat, mouse Bers 2010 10 Ca2+ release in permeabilized CM Sticky FKBP12.6 reduces Ca2+ leak but does FKBP12.6D37S TG x CaMKII TG Mouse Maier 2010 23 not rescue contractile or structural pathology of CaMKII TG Nitrosylation and oxidation affects RyR2 NO inhibits RyR2 Po, SH-group reduction SR vesicles lipid bilayer, Rabbit Meszaros 1997 36 reverses this effect l-arginine, NOS inhibitors Little oxidation had no effect, medium oxidation SR vesicles, lipid bilayer, Rabbit Meissner 2001 30 increases in Po, strong oxidation inactivates number of free SH groups RyR2; no role of S-nitrosylation and tyrosine nitration HF associated with (antioxidant-sensitive) Pacing-induced HF with or without Dog Matsuzaki 2005 34 loss of SH groups + Ca2+ leak antioxidant, SR vesicles Nos1–/– mice: less RyR2 nitrosylation, Nos1–/– mice, Nos3–/– mice, or DKO Mouse Hare 2007 32 more SH oxidation, increased Po GSNO but not NO directly affects RyR2 SR vesicles, lipid bilayer, different pO2 Dog Meissner 2008 31 in a pO2-dependent manner Binding of FKBP12.6 to RyR2 is redox sensitive

H2O2 and diamide reduce FKBP12.6 binding SR vesicles, FKB-P12.6 binding Dog Lai 2007 33 by 25% and 50%, respectively, JTV 519 with or without DTT, H2O2, diamide did not rescue SH oxidation in Nos–/– mice was not Nos1–/– mice, Nos3–/– mice, Mouse Hare 2007 32 associated with altered FKBP12.6 or DKO binding to RyR2

DKO, double-knockout mice; DTT, dithiothreitol (reducing agent); TG, transgenic mice.

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did not improve cardiac pathology in a transgenic mouse model of heart failure (with increased Ca2+ leak), arguing against the universal role of leaky RyR2 in the pathophysiology of heart failure. Finally, others (24) could neither reproduce the effect of FKBP12.6 dissociation on RyR2 open probability nor the proarrhythmic phenotype of Fkbp12.6-knockout mice observed by the Marks group (16).

Evidence from gene-targeted, phospho-mutant RyR2 mouse models In this confusing state of the RyR2 litera- ture, one would expect “clean” knockin mouse models to finally provide a definite answer. Yet, this is only partially the case, as shown by two studies from the Marks laboratory published in this issue of the JCI (25, 26). The two papers report on two new mouse models: one in which the S2808 site was mutated to a non-phosphorylatable alanine (S2808A; ref. 25), and another in Figure 2 which S2808 was replaced by aspartic acid The leaky RyR2 hypothesis: phosphorylation and oxidation of RyR2 in the center of a vicious (S2808D), a “phosphomimetic” amino circle in heart failure. According to Marks and colleagues, catecholamines control excitation- acid (26). The expectation was that S2808A contraction coupling gain not only at the level of LTCCs and PLB, but also at the level of RyR2 by mice would be less sensitive to the force- phosphorylating it at S2808. The latter reduces RyR2 affinity for the stabilizing accessory protein, increasing acute effects of isoprenaline FKBP12.6, and increases its open probability. In heart failure, sustained catecholamine stimula- (blunted fight or flight response) but also tion leads to hyperphosphorylation, leaky RyR2, spontaneous Ca2+ release and, via NCX, spon- taneous depolarizations. The new data presented in this issue of the JCI (25, 26) now suggest protected from adverse long-term conse- that phosphorylation at S2808 alone does not suffice to dissociate FKBP12.6 but that it needs quences of b-adrenergic stimulation, such oxidation of the channel plus phosphorylation. The level of oxidizing ROS is commonly increased as cardiac dilatation and dysfunction (as in heart failure and, importantly, the increased Ca2+ leak from RyR2 (in consequence of oxidation seen in mice lacking phosphatase inhibi- and phosphorylation) further increases ROS production, e.g., from mitochondria. This consti- tor-1; ref. 27). Conversely, S2808D mice tutes a classical vicious circle. Black arrows and lettering indicate basic excitation-contraction would be expected to exhibit spontaneous coupling; red arrows indicate changes under chronic catecholamine stimulation and heart failure. FKBP12.6 depletion from the RyR2 com- The red dotted line indicates Ca2+ leak. Adapted with permission from Nature (35). plex, initial hypercontractility (because of facilitated Ca2+ release from the SR), spon- taneous development of a cardiomyopath- that the effect in WT myocytes is exclu- data on isolated dog SR that reported more ic phenotype, and exaggerated pathology sively due to increased SR Ca2+ loading (via than 80% occupancy (15), suggest that under chronic stress. The results obtained PLB phosphorylation). This experiment a change of RyR2 affinity for FKBP12.6 by Marks and colleagues almost exactly also shows that the contribution of RyR would not affect more than a minority of matched these expectations and go beyond cannot be judged without controlling for RyR2. (c) Another exciting finding by the (25, 26). S2808A mice also exhibited blunt- SR Ca2+ load. Others have studied isolated Marks group (16), namely that CPVT-asso- ed chronotropic responses (i.e., blunted RyR2 preparations and observed that not ciated mutations in RyR2 are associated acceleration of the heart rate) to isoprena- only phosphorylation at S2808, but also with decreased FKBP12.6 binding, has line and reduced exercise capacity, suggest- dephosphorylation, increased its open also been challenged by reports of unal- ing that RyR2 phosphorylation at S2808 probability (9, 11). Several groups have tered or even increased binding affinity of regulates not only contractile force but failed to reproduce the effect observed by mutant RyR2 channels for FKBP12.6 (17). heart rate too and that it is a critical deter- Marks and colleagues (2) of PKA phos- (d) Several reports support the idea that minant of physical fitness. The S2808D phorylation at S2808 or mutations of this removal of FKBP12.6 from the RyR2 com- experiments also enlarge the picture by site on FKBP12.6 binding (2, 10, 12, 13), plex results in increased open probability, showing that the b-blockers metoprolol but others have (14). Moreover, a recent spark frequency, and arrhythmias (18, 19) and carvedilol, while effective in infarcted study by the Bers group (10) reported very and thus support this aspect of the Marks WT mice, had no effect in S2808D mice. In low FKBP12.6 concentrations in rodent theory (2, 16, 20). Conversely, overexpres- contrast, the Ca2+ release channel–stabiliz- cardiomyocytes, resulting in only 10%–20% sion of FKBP12.6 (or sticky mutants) was ing drug S107 stabilized FKBP12.6 binding of RyR2 being occupied by FKBP12.6. accompanied by a reduced propensity for to RyR2 and ameliorated cardiac dysfunc- These data, although in contrast to earlier cardiac arrhythmia (21–23). However, this tion in S2808D mice. Taken together, the

The Journal of Clinical Investigation http://www.jci.org Volume 120 Number 12 December 2010 4201 commentaries new results from the Marks group strongly the entire dose range (exhibiting a clear ferent protocols and laboratories and are support a key role for phosphorylation of plateau at approximately 0.3 mg/kg/min rarely explicitly reported (e.g., some inves- RyR2 S2808 in b-adrenergic regulation in both WT and S2808A mice). Moreover, tigators use ascorbic acid in their Tyrode’s of cardiac force and frequency, in setting a saturating concentration of isoprenaline solution, others do not). Thus, the idea maximal exercise capacity, in determining (100 nmol/l) exerted less than a maximal that different redox states of RyR2 account cardiac responses to chronic stress, and in force response in free-running Langen- for at least some of the published discrep- the protective effect of b-blockers. dorff-perfused S2808A mice. Marks and ancies is appealing. Based on experiments colleagues therefore propose (25) that presented in Figure 1D of ref. 25, Marks The new findings in the context the differences between their results and and colleagues propose that oxidation of the published state of the art? those of the Houser group (at 10-fold of RyR2 sensitizes the channel for PKA Unfortunately, these new results from the lower concentrations) were due to the fact phosphorylation at S2808 and destabi- Marks laboratory (25, 26) are in striking that Houser’s group used paced Langen- lizes FKBP12.6 binding. Conversely, under contrast to studies from the groups of dorff-hearts (480 beats/min). However, as non-oxidizing conditions, PKA may not Valdivia and Houser in an independently demonstrated in Figure 3 of ref. 25, pac- induce FKBP12.6 dissociation, and this generated S2808A knockin mouse line ing (at 600 beats/min) reduced but did not might then explain why others did not (28, 29). These mice not only displayed abolish the differences between WT and find it. The spontaneous cardiomyopathic normal heart dimensions before and after S2808A mice. The Marks group also argues phenotype of S2808D mice observed by aortic banding but also normal myocyte that in the Valdivia study (28) S2808A Marks and colleagues was accompanied shortening and Ca2+ transients, normal mouse myocytes did not react exactly as by increased S-nitrosylation and more responses to isoprenaline, and, interest- WT myocytes in response to isoprenaline pronounced SH oxidation (Figure 2B ingly, normal PKA-catalyzed incorpora- (Figure 5C in ref. 28). Whereas this is for- of ref. 26). This heart failure–associated tion of radioactive phosphate into RyR2, mally true, the slightly (but statistically increase in oxidation could then explain despite effective elimination of S2808 significantly) higher Ca2+ transient in WT why it took 6 months for S2808D mice to phosphorylation. The latter result sup- myocytes at 3 Hz does not strike as a rel- exhibit significant loss of FKBP12.6 from ports data suggesting that S2030 is the evant difference to the author of this com- the RyR2 complex (Figure 2, A and B, of dominant PKA phosphorylation site on mentary. Finally, the Marks group shows ref. 26), despite the proposed “hyperphos- RyR2 and that S2808 is of minor quanti- in the second paper (26) that Ca2+ spark phorylation state” of RyR2 being present tative relevance (8). frequency in 6- to 8-month-old S2808D throughout life. The hypothesis derived by How can one reconcile these different mouse myocytes was larger than in WT, Marks and colleagues from these results is results with apparently identical mutant despite lower SR Ca2+ load. It is difficult to that both oxidative stress (as commonly mouse lines? Marks and colleagues pres- explain these data by an isolated change in seen in heart failure; ref. 34) and PKA ent two major lines of reasoning (25, 26). RyR2 open probability, because such situ- phosphorylation cooperate to destabilize The first one is that the other studies used ation automatically leads to a new equilib- the RyR2-FKBP12.6 complex (Figure 2 doses/concentrations of isoprenaline and rium at a lower SR load (4). of this commentary). The increased Ca2+ experimental conditions (pacing) that A second hypothesis to explain the con- leak then promotes ROS generation (e.g., obscured differences in sensitivity. This troversy relates to redox modifications of from mitochondria) and thus accelerates explanation is based on a key experiment RyR2. It is well known and undisputed the vicious circle (Figure 2 of this com- performed by the Marks group (25), show- that RyR1 (the skeletal isoform) and RyR2 mentary). This elegant hypothesis could ing that low doses of isoprenaline (2 mg/kg) are redox responsive (Tables 1 and 2). RyR1 indeed explain some of the experimental had markedly smaller chronotropic and contains 101 thiols/subunit, and their oxi- discrepancies, particularly those in vitro. inotropic effects in S2808A than in WT dation (e.g., by ROS) increases RyR1 open It is more difficult to assume that it can mice but a 1,000-fold higher dose exerted probability at low/moderate levels of oxi- explain discrepancies between experiments similar maximal effects (Figure 1 of ref. dation (and irreversibly inactivates it at in whole heart homogenates, intact cells/ 25). Indeed, the Valdivia and the Houser high levels; ref. 30). RyR2 is also subject to hearts, and entire animals. studies used saturating concentrations/ oxidation and S-nitrosylation (likely via doses of isoprenaline in vitro (1 mmol/l) S-nitrosoglutathione) in a pO2-dependent Conclusion and in vivo (2 mg/kg), respectively (28, 29). manner (31). Recent work suggests com- Although the controversies surrounding However, dosing alone cannot account petition between NOS1/NO-dependent RyR2 phosphorylation are sure to con- for the strikingly different observations S-nitrosylation and ROS-dependent tinue, the means to directly test evolving for two reasons. First, the Houser group SH oxidation, with the latter leading to hypotheses are at hand. Direct head-to- also tested the effects of isoprenaline on increased leakiness of the channel (32). head comparisons of available animal Langendorff-perfused mouse hearts at 10 One report indicates that SH oxidation models and other reagents in multiple nmol/l, a concentration that is clearly not reduces the affinity of RyR2 for FKBP12.6 laboratories and the further development saturating, and observed essentially identi- (33), but another shows that the level of of ever-more refined reagents will help to cal effects in S2808A and WT hearts (29). FKBP12.6 associated with RyR2 is normal clarify this fundamental aspect of cardio- Second, Figure 3 of ref. 25 shows, some- despite its increased oxidation in Nos–/– vascular physiology. what in contrast to their own findings mice (32). In any case, there is little doubt presented in Figure 1 in the same paper , that redox modifications can profoundly Acknowledgments that the contractile force (dP/dt) response affect RyR2 function. Moreover, redox The work of the author is supported by was reduced in S2808A mouse hearts over conditions can easily differ between dif- grants from the German Research Founda-

4202 The Journal of Clinical Investigation http://www.jci.org Volume 120 Number 12 December 2010 commentaries tion (DFG FOR 604, Es 88/10-1), the Euro- 10. Guo T, et al. Kinetics of FKBP12.6 binding to eling and heart failure [published online ahead ryanodine receptors in permeabilized cardiac of print August 24, 2010]. J Mol Cell Cardiol. doi: pean Union (EUGeneHeart, Angioscaff), myocytes and effects on Ca sparks. Circ Res. 2010; 10.1016/j.yjmcc.2010.08.016. and the German Heart Foundation. 106(11):1743–1752. 24. Xiao B, et al. Functional consequence of protein 11. Terentyev D, Viatchenko-Karpinski S, Gyorke I, kinase A-dependent phosphorylation of the car- Address correspondence to: T. Eschenhagen, Terentyeva R, Gyorke S. Protein phosphatases diac ryanodine receptor: sensitization of store decrease sarcoplasmic reticulum calcium content overload-induced Ca2+ release. J Biol Chem. 2007; Department of Experimental Pharmacolo- by stimulating calcium release in cardiac myocytes. 282(41):30256–30264. gy and Toxicology, Cardiovascular Research J Physiol. 2003;552(pt 1):109–118. 25. Shan J, et al. Phosphorylation of the ryano- Center Hamburg, University Medical Cen- 12. Stange M, Xu L, Balshaw D, Yamaguchi N, Meiss- dine receptor mediates the cardiac fight or ner G. Characterization of recombinant skeletal flight response in mice. J Clin Invest. 2010; ter Hamburg Eppendorf, Martini-strasse muscle (Ser-2843) and (Ser-2809) 120(12):4388–4398. 52, 20246 Hamburg, Germany. Phone: ryanodine receptor phosphorylation mutants. 26. Shan J, et al. Role of chronic ryanodine receptor 49.40.74105.2180; Fax: 49.40.74105.4876; J Biol Chem. 2003;278(51):51693–51702. phosphorylation in heart failure and β-adrener- 13. Xiao B, Sutherland C, Walsh MP, Chen SR. Protein gic receptor blockade in mice. 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