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Intracellular Zinc Modulates Cardiac Ryanodine Receptor-mediated Calcium Release*

  • Jason Woodier
    Affiliations
    From the School of Medicine, University of St. Andrews, St. Andrews KY16 9TF, United Kingdom and
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  • Richard D. Rainbow
    Affiliations
    the Department of Cardiovascular Sciences, University of Leicester, Clinical Sciences Wing, Glenfield General Hospital, Leicester LE3 9QP, United Kingdom
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  • Alan J. Stewart
    Affiliations
    From the School of Medicine, University of St. Andrews, St. Andrews KY16 9TF, United Kingdom and
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  • Samantha J. Pitt
    Correspondence
    Supported by a Royal Society of Edinburgh Biomedical Research Fellowship. To whom correspondence should be addressed: School of Medicine, University of St. Andrews, Medical and Biological Sciences Bldg., North Haugh, St. Andrews, Fife, KY6 9TF, UK. Tel.: 44-1334-463516;
    Affiliations
    From the School of Medicine, University of St. Andrews, St. Andrews KY16 9TF, United Kingdom and
    Search for articles by this author
  • Author Footnotes
    * This work was supported by University of St. Andrews, Tenovus Scotland Grant T14/35 (to S. J. P.), British Heart Foundation Grant FS/14/69/31001 (to S. J. P.), a grant from the John and Lucille van Geest Cardiovascular Diseases Research Fund (to the University of Leicester and R. D. R.), and Biotechnology and Biological Sciences Research Council Grant BB/J006467/1 (to A. J. S.). The authors declare that they have no conflicts of interest with the contents of this article.
Open AccessPublished:June 03, 2015DOI:https://doi.org/10.1074/jbc.M115.661280
      Aberrant Zn2+ homeostasis is a hallmark of certain cardiomyopathies associated with altered contractile force. In this study, we addressed whether Zn2+ modulates cardiac ryanodine receptor gating and Ca2+ dynamics in isolated cardiomyocytes. We reveal that Zn2+ is a high affinity regulator of RyR2 displaying three modes of operation. Picomolar free Zn2+ concentrations potentiate RyR2 responses, but channel activation is still dependent on the presence of cytosolic Ca2+. At concentrations of free Zn2+ >1 nm, Zn2+ is the main activating ligand, and the dependence on Ca2+ is removed. Zn2+ is therefore a higher affinity activator of RyR2 than Ca2+. Millimolar levels of free Zn2+ were found to inhibit channel openings. In cardiomyocytes, consistent with our single channel results, we show that Zn2+ modulates both the frequency and amplitude of Ca2+ waves in a concentration-dependent manner and that physiological levels of Zn2+ elicit Ca2+ release in the absence of activating levels of cytosolic Ca2+. This highlights a new role for intracellular Zn2+ in shaping Ca2+ dynamics in cardiomyocytes through modulation of RyR2 gating.
      Background: In heart failure, the release of calcium becomes erratic leading to the generation of arrhythmias. Dysregulated Zn2+ homeostasis occurs in chronic heart failure.
      Results: Zn2+ can directly activate RyR2, removing the dependence of Ca2+ for channel activation.
      Conclusion: Zn2+ shapes Ca2+ dynamics by directly interacting with and modulating RyR2 function.
      Significance: This highlights a new role for Zn2+ in cardiac excitation-contraction coupling.

      Introduction

      In cardiac muscle, the intracellular signal that triggers muscle contraction is thought to be a transient rise in intracellular Ca2+ that leads to the opening of Ca2+ release channels called type 2 ryanodine receptors (RyR2)
      The abbreviations used are: RyR2
      type 2 ryanodine receptor
      SR
      sarcoplasmic reticulum
      BAPTA
      2,2′-(ethylenedioxy)dianiline-N,N,N′,N′-tetraacetic acid
      TPEN
      N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine
      ZnPy
      zinc pyrithione
      ANOVA
      analysis of variance.
      on the sarcoplasmic reticulum (SR). The resulting release of Ca2+ into the cytosol causes movement of contractile myofibrils leading to cell contraction. Damaging changes in Ca2+ homeostasis are associated with heart failure, conduction abnormalities, and contractile dysfunction. Abnormal RyR2 function is recognized as an important component in the etiology of such disease states (
      • Belevych A.E.
      • Radwański P.B.
      • Carnes C.A.
      • Györke S.
      “Ryanopathy”: causes and manifestations of RyR2 dysfunction in heart failure.
      ,
      • Eisner D.
      • Bode E.
      • Venetucci L.
      • Trafford A.
      Calcium flux balance in the heart.
      • Kranias E.G.
      • Bers D.M.
      Calcium and cardiomyopathies.
      ).
      Recently, there has been much interest in the role of Zn2+ as an intracellular signaling molecule (
      • Maret W.
      Crosstalk of the group IIa and IIb metals calcium and zinc in cellular signaling.
      ,
      • Fukada T.
      • Yamasaki S.
      • Nishida K.
      • Murakami M.
      • Hirano T.
      Zinc homeostasis and signaling in health and diseases.
      • Yamasaki S.
      • Sakata-Sogawa K.
      • Hasegawa A.
      • Suzuki T.
      • Kabu K.
      • Sato E.
      • Kurosaki T.
      • Yamashita S.
      • Tokunaga M.
      • Nishida K.
      • Hirano T.
      Zinc is a novel intracellular second messenger.
      ). Like Ca2+, intracellular Zn2+ is heavily buffered (
      • Colvin R.A.
      • Holmes W.R.
      • Fontaine C.P.
      • Maret W.
      Cytosolic zinc buffering and muffling: their role in intracellular zinc homeostasis.
      ), and cellular Zn2+ homeostasis requires mechanisms that tightly control the uptake, storage, and distribution of Zn2+. This is achieved through the coordinated actions of metallothionein proteins and Zn2+ transporters (
      • Krezel A.
      • Maret W.
      Zinc-buffering capacity of a eukaryotic cell at physiological pZn.
      ,
      • Mocchegiani E.
      • Giacconi R.
      • Malavolta M.
      Zinc signalling and subcellular distribution: emerging targets in type 2 diabetes.
      • Lichten L.A.
      • Cousins R.J.
      Mammalian zinc transporters: nutritional and physiologic regulation.
      ).
      In cardiomyocytes, the resting intracellular Zn2+ concentration is reported to be at picomolar levels (∼100 pm) (
      • Chabosseau P.
      • Tuncay E.
      • Meur G.
      • Bellomo E.A.
      • Hessels A.
      • Hughes S.
      • Johnson P.R.
      • Bugliani M.
      • Marchetti P.
      • Turan B.
      • Lyon A.R.
      • Merkx M.
      • Rutter G.A.
      Mitochondrial and ER-targeted eCALWY probes reveal high levels of free Zn2+.
      ,
      • Turan B.
      • Fliss H.
      • Désilets M.
      Oxidants increase intracellular free Zn2+ concentration in rabbit ventricular myocytes.
      ). During cardiac excitation-contraction coupling, intracellular Zn2+ concentrations are altered, and spatiotemporal fluctuations in free Zn2+ levels, including both Zn2+ transients and Zn2+ sparks, are suggested to be remarkably similar to those previously shown for Ca2+ (
      • Tuncay E.
      • Bilginoglu A.
      • Sozmen N.N.
      • Zeydanli E.N.
      • Ugur M.
      • Vassort G.
      • Turan B.
      Intracellular free zinc during cardiac excitation-contraction cycle: calcium and redox dependencies.
      ). Other groups suggest that Zn2+ can permeate through the L-type channel with a greater affinity than Ca2+ but with a much lower permeability leading to a reduction in the inward current (
      • Alvarez-Collazo J.
      • Díaz-García C.M.
      • López-Medina A.I.
      • Vassort G.
      • Alvarez J.L.
      Zinc modulation of basal and β-adrenergically stimulated L-type Ca2+ current in rat ventricular cardiomyocytes: consequences in cardiac diseases.
      ,
      • Atar D.
      • Backx P.H.
      • Appel M.M.
      • Gao W.D.
      • Marban E.
      Excitation-transcription coupling mediated by zinc influx through voltage-dependent calcium channels.
      ). It has also been suggested that the SR can function as an intracellular store for Zn2+ alongside Ca2+ (
      • Yamasaki S.
      • Sakata-Sogawa K.
      • Hasegawa A.
      • Suzuki T.
      • Kabu K.
      • Sato E.
      • Kurosaki T.
      • Yamashita S.
      • Tokunaga M.
      • Nishida K.
      • Hirano T.
      Zinc is a novel intracellular second messenger.
      ,
      • Tuncay E.
      • Bilginoglu A.
      • Sozmen N.N.
      • Zeydanli E.N.
      • Ugur M.
      • Vassort G.
      • Turan B.
      Intracellular free zinc during cardiac excitation-contraction cycle: calcium and redox dependencies.
      ).
      Importantly aberrant Zn2+ homeostasis has been shown to be associated with cardiomyopathy including chronic heart failure, and myocardial damage as a result of dysregulated intracellular Ca2+ release, reduced cardiac contractility, and significantly prolonged rises of systolic Ca2+ (
      • Reuter H.
      • Grönke S.
      • Adam C.
      • Ribati M.
      • Brabender J.
      • Zobel C.
      • Frank K.F.
      • Wippermann J.
      • Schwinger R.H.
      • Brixius K.
      • Müller-Ehmsen J.
      Sarcoplasmic Ca2+ release is prolonged in nonfailing myocardium of diabetic patients.
      • Kleinfeld M.
      • Stein E.
      Action of divalent cations on membrane potentials and contractility in rat atrium.
      ,
      • Ciofalo F.R.
      • Thomas Jr., L.J.
      The effects of zinc on contractility, membrane potentials, and cation content of rat atria.
      • Kalfakakou V.P.
      • Evangelou A.M.
      • Benveniste J.
      • Arnoux B.
      The effects of Zn2+ on guinea pig isolated heart preparations.
      ). The potential role of Zn2+ in shaping intracellular Ca2+ release and regulating intracellular Ca2+ dynamics in heart however, is poorly characterized. Here we show that Zn2+ is a potent regulator of SR Ca2+ release through modulation of RyR2 channel function and that Zn2+ plays a key role in shaping intracellular Ca2+ dynamics important in cardiac excitation-contraction coupling.

      Discussion

      In this study, we reveal that cytosolic Zn2+ is a high affinity activator of RyR2, effective at low picomolar concentrations. Lifetime analysis of RyR2 reveals unique and distinctive gating characteristics dependent on the concentration of cytosolic Zn2+. When Zn2+ levels are in the range 100 pm to 1 nm, RyR2 activation occurs by sensitization of the channel to cytosolic Ca2+. When the concentration of Zn2+ is elevated above 1 nm, the effect of Zn2+ overrides the influence of cytosolic Ca2+, and removal of activating levels of cytosolic Ca2+ no longer influences channel Po. When Zn2+ is elevated to toxic levels (in the millimolar range), it inhibits RyR2 channel function. Further evidence for the modulation of RyR2 function by Zn2+ in isolated permeabilized cardiomyocytes was acquired through measurement of Ca2+ waves. In these recordings, spontaneous Ca2+ waves were markedly increased in both frequency and amplitude in a Zn2+ concentration-dependent manner (between 100 pm and 10 nm Zn2+). Removal of Ca2+ by chelation with 1 mm BAPTA did not abolish Ca2+ waves in the presence of Zn2+, showing that Zn2+ can directly influence Ca2+ dynamics. These findings suggest an important pathophysiological consequence of dysregulated Zn2+ homeostasis, whereby even a small increase in RyR2 open probability during diastole can have major consequences for normal cardiac function and the generation of arrhythmias (
      • Bers D.M.
      Calcium cycling and signaling in cardiac myocytes.
      ).
      It is well established that micromolar concentrations of cytosolic Ca2+ are required to activate RyR2 (
      • Rousseau E.
      • Smith J.S.
      • Henderson J.S.
      • Meissner G.
      Single channel and 45Ca2+ flux measurements of the cardiac sarcoplasmic reticulum calcium channel.
      ,
      • Xu L.
      • Mann G.
      • Meissner G.
      Regulation of cardiac Ca2+ release channel (ryanodine receptor) by Ca2+, H+, Mg2+ and adenine nucleotides under normal and simulated ischemic conditions.
      ). The potentiation of RyR2 activity by physiological concentrations of Zn2+ that we now show suggests that Zn2+ plays a key role in shaping intracellular Ca2+ responses in cardiac muscle. Tight regulation of intracellular Zn2+ will therefore be required for the controlled release of Ca2+ from SR stores because very small changes in the concentration of free Zn2+ will modify the opening of RyR2 channels in response to very small rises in cytosolic Ca2+, and this may cause channels to become leaky during diastole. Under pathophysiological conditions, where Zn2+ homeostasis is disturbed and levels of Zn2+ may reach >1 nm, we propose that Zn2+ becomes the main activating ligand of RyR2. Under these conditions, RyR2 starts to gate in exceptionally long open states. The ability of 1 nm Zn2+ to activate RyR2 reveals that Zn2+ is a high affinity activator of RyR2 displaying approximately 3 orders of magnitude higher affinity for RyR2 than Ca2+. This alters the current model of cardiac excitation-contraction coupling where it is accepted that a transient changes in the concentration of intracellular Ca2+ is the only trigger for SR Ca2+ release and reveals a new role for Zn2+ in shaping intracellular calcium responses in cardiac muscle.
      Determination of the exact levels of intracellular Zn2+ under physiological and pathophysiology conditions is an ongoing area of research, but the development of more sensitive Zn2+ probes that are able to measure low concentrations of free Zn2+ in single living cells is rapidly advancing (
      • Vinkenborg J.L.
      • Nicolson T.J.
      • Bellomo E.A.
      • Koay M.S.
      • Rutter G.A.
      • Merkx M.
      Genetically encoded FRET sensors to monitor intracellular Zn2+ homeostasis.
      ). At reported pathophysiological levels of cytosolic Zn2+ (∼30 nm) (
      • Sensi S.L.
      • Paoletti P.
      • Bush A.I.
      • Sekler I.
      Zinc in the physiology and pathology of the CNS.
      • Aizenman E.
      • Stout A.K.
      • Hartnett K.A.
      • Dineley K.E.
      • McLaughlin B.
      • Reynolds I.J.
      Induction of neuronal apoptosis by thiol oxidation.
      ,
      • Li Y.
      • Maret W.
      Transient fluctuations of intracellular zinc ions in cell proliferation.
      • Ayaz M.
      • Turan B.
      Selenium prevents diabetes-induced alterations in [Zn2+]i and metallothionein level of rat heart via restoration of cell redox cycle.
      ), we show that increases in RyR2 activity are the result of large increases in the lifetime duration of the open channel state rather than an increase in the frequency of openings and this is characteristic of a Ca2+-independent component of channel gating. In the presence of ≥10 nm free Zn2+, we show that chelation of cytosolic Ca2+ is no longer able to close RyR2 channels. This is because there is sufficient Zn2+ present to directly open the channel. The balance of intracellular Zn2+ is therefore crucial in switching RyR2 channels from a “Ca2+-dependent” to a “Ca2+-independent” mode of gating.
      Our mechanistic approach has for the first time revealed that Zn2+ is a potent activator of RyR2. Previous studies in which cardiac muscle [3H]ryanodine binding assays were used to assess the effects of Zn2+ on RyR2 function failed to see any effect of Zn2+ between 10 nm and 1 μm in the presence of 50 μm activating Ca2+ (
      • Wang H.
      • Wei Q.Q.
      • Cheng X.Y.
      • Chen K.Y.
      • Zhu P.H.
      Inhibition of ryanodine binding to sarcoplasmic reticulum vesicles of cardiac muscle by Zn2+ ions.
      ). This is not surprising because our data now reveal that in the presence of peak levels of Ca2+, the effect of Zn2+ on channel Po is masked. Only by lifetime analysis of our single channel data could we reveal that the mode of channel gating in the presence of Zn2+ (>1 nm) was altered and that this type of gating was consistent with Ca2+-independent openings. Our single channel data also provide the first evidence that when the concentration of Zn2+ is elevated above 1 nm, RyR2 becomes directly activated by Zn2+, and the dependence of Ca2+ on channel activation is abolished.
      It is notable that previous studies have shown a slow accumulation of intracellular Zn2+ following prolonged exposure of cardiomyocytes to micromolar levels of extracellular Zn2+ and that this results in a reduced SR Ca2+ load (
      • Yi T.
      • Vick J.F.
      • Vecchio M.F.
      • Begin K.F.
      • Bell S.F.
      • Delay R.F.
      • Palmer B.M.
      Identifying cellular mechanisms of zinc-induced relaxation in isolated cardiomyocytes.
      ). One plausible explanation for these findings, based on our current data, is that an elevation in intracellular Zn2+ removes the Ca2+ dependence of RyR2 causing channels to gate in exceptionally long-lived open states. Further evidence for this is demonstrated in intact cardiomyocytes where, in a 100 pm Zn2+-Tyrode's solution, the caffeine-induced elevation in intracellular Ca2+ was potentiated only after application of 10 μm ZnPy. Potentially this will lead to inappropriate Ca2+ release from the SR and likely result in reduced SR Ca2+ load. Such a mechanism would be particularly problematic under conditions where levels of intracellular Zn2+ are chronically elevated, including diabetes (
      • Ayaz M.
      • Turan B.
      Selenium prevents diabetes-induced alterations in [Zn2+]i and metallothionein level of rat heart via restoration of cell redox cycle.
      ) and certain models of dystrophy (
      • Crawford A.J.
      • Bhattacharya S.K.
      Excessive intracellular zinc accumulation in cardiac and skeletal muscles of dystrophic hamsters.
      ). In line with previous studies (
      • Wang H.
      • Wei Q.Q.
      • Cheng X.Y.
      • Chen K.Y.
      • Zhu P.H.
      Inhibition of ryanodine binding to sarcoplasmic reticulum vesicles of cardiac muscle by Zn2+ ions.
      ,
      • Xie H.
      • Chen K.Y.
      • Zhu P.H.
      Effect of Zn2+ ions on ryanodine binding to sarcoplasmic reticulum of striated muscles in the presence of pyrithione.
      ), we also demonstrated an inhibitory action of Zn2+ on RyR2 gating when cytosolic Zn2+ is raised to nonphysiological and toxic millimolar concentrations. It is possible that the inhibitory action of Zn2+ on RyR2 may be a consequence of Zn2+ binding to the low affinity unspecific divalent inhibitory site of RyR2 (
      • Laver D.R.
      • Baynes T.M.
      • Dulhunty A.F.
      Magnesium inhibition of ryanodine-receptor calcium channels: evidence for two independent mechanisms.
      ,
      • Diaz-Sylvester P.L.
      • Porta M.
      • Copello J.A.
      Modulation of cardiac ryanodine receptor channels by alkaline earth cations.
      ).
      Aberrant Zn2+ homeostasis has been shown to be associated with cardiomyopathy (
      • Little P.J.
      • Bhattacharya R.
      • Moreyra A.E.
      • Korichneva I.L.
      Zinc and cardiovascular disease.
      ,
      • Sandstead H.H.
      Requirements and toxicity of essential trace elements, illustrated by zinc and copper.
      • Alexanian I.
      • Parissis J.
      • Farmakis D.
      • Athanaselis S.
      • Pappas L.
      • Gavrielatos G.
      • Mihas C.
      • Paraskevaidis I.
      • Sideris A.
      • Kremastinos D.
      • Spiliopoulou C.
      • Anastasiou-Nana M.
      • Lekakis J.
      • Filippatos G.
      Clinical and echocardiographic correlates of serum copper and zinc in acute and chronic heart failure.
      ), but the underlying mechanism of how Zn2+ contributes to cardiac pathology is unknown. Chronic oxidative stress is a major contributor in the pathophysiology of heart failure and under these conditions intracellular levels of Zn2+ increase by as much as 30-fold (
      • Ayaz M.
      • Turan B.
      Selenium prevents diabetes-induced alterations in [Zn2+]i and metallothionein level of rat heart via restoration of cell redox cycle.
      ). This increase in Zn2+ is primarily the result of altered metallothionein function, which perturbs its binding of Zn2+, resulting in raised levels (
      • Maret W.
      Oxidative metal release from metallothionein via zinc-thiol/disulfide interchange.
      ,
      • Maret W.
      Metallothionein/disulfide interactions, oxidative stress, and the mobilization of cellular zinc.
      ). Also, recently, a polymorphism in a human gene coding for a member of the metallothionein family has been discovered, and individuals with diabetes who carry this polymorphism have been shown to be more likely to develop cardiovascular complications including chronic heart failure and myocardial damage (
      • Giacconi R.
      • Bonfigli A.R.
      • Testa R.
      • Sirolla C.
      • Cipriano C.
      • Marra M.
      • Muti E.
      • Malavolta M.
      • Costarelli L.
      • Piacenza F.
      • Tesei S.
      • Mocchegiani E.
      +647 A/C and +1245 MT1A polymorphisms in the susceptibility of diabetes mellitus and cardiovascular complications.
      ). Diabetic cardiomyopathy is characterized by dysregulation of intracellular Ca2+ release, which consequently reduces cardiac contractility and significantly prolongs the rise of systolic Ca2+ leading to chronic heart failure (
      • Reuter H.
      • Grönke S.
      • Adam C.
      • Ribati M.
      • Brabender J.
      • Zobel C.
      • Frank K.F.
      • Wippermann J.
      • Schwinger R.H.
      • Brixius K.
      • Müller-Ehmsen J.
      Sarcoplasmic Ca2+ release is prolonged in nonfailing myocardium of diabetic patients.
      ). The controlled release of Ca2+ from the SR during cardiac excitation-contraction coupling is known to govern contractility of the heart. RyR2 plays a fundamental role as the main pathway for the release of Ca2+ and drives cellular contraction. Consequently dysfunction in the release of Ca2+ and modulatory influences that control RyR2 function are identified as contributory to the pathophysiology of heart failure and fatal cardiac arrhythmias (
      • Belevych A.E.
      • Radwański P.B.
      • Carnes C.A.
      • Györke S.
      “Ryanopathy”: causes and manifestations of RyR2 dysfunction in heart failure.
      ). The presented study therefore provides a mechanistic explanation as to how raised levels of intracellular Zn2+ may contribute to the progression of contractile dysfunction and heart failure through the alteration of RyR2 gating, which will result in perturbed Ca2+ release.
      A simple model to explain how cytosolic Zn2+ modulates RyR2 gating is shown in Fig. 10. We suggest that under physiological conditions, cytosolic Zn2+ is able to fine-tune the release of Ca2+ from the SR while in parallel enabling flexibility and control of SR Ca2+ dynamics mediated through RyR2. Under pathophysiological conditions, where Zn2+ homeostasis is disturbed and levels of Zn2+ may reach >1 nm, RyR2 starts to gate in exceptionally long open states, which leads to very high Po potentially leading to arrhythmia. In this conformation, RyR2 is uncoupled from the usual regulatory effects of cytosolic Ca2+ and RyR2 channels are now activated by solely by Zn2+.
      Figure thumbnail gr10
      FIGURE 10.Proposed model of how Zn2+ regulates RyR2-mediated Ca2+ release. When 10 μm cytosolic Ca2+ is the sole ligand, channel openings are brief, and the channel gates with a low Po. Addition of 1 nm Zn2+ to the cytosolic face of the channel causes an increase in the channel Po, and the channel gates with high frequency openings. In both of these conditions, RyR2 gating is regulated by cytosolic Ca2+, and lowering the concentration of free Ca2+ to subactivating levels (<10 nm) reduces RyR2 Po to 0. If the Zn2+ concentration is elevated above 1 nm, RyR2 becomes uncoupled from the usual regulatory effects of cytosolic Ca2+, and Zn2+ becomes the sole activating ligand.
      Our study reveals that RyR2-mediated Ca2+ homeostasis is intimately related to intracellular Zn2+ levels and provides a mechanistic explanation as to how altered Zn2+ homeostasis can modulate cardiac RyR2 function. We reveal that Zn2+ is a high affinity activator of RyR2 which challenges our understanding of cardiac excitation-contraction coupling. We suggest that under normal physiological conditions, intracellular Zn2+ is essential in fine-tuning the release of Ca2+ from the SR. Pathological perturbations in Zn2+ homoeostasis will lead to inappropriate release of Ca2+ as observed in certain cardiac abnormalities including heart failure and fatal arrhythmias.

      Author Contributions

      J. W. and R. D. R. performed experiments and analyzed data; A. J. S. designed the experiments and contributed toward writing the manuscript; and S. J. P. designed the experiments, performed experiments, analyzed data, wrote the manuscript, and supervised the project. All authors discussed the results and implications and commented on the manuscript at all stages.

      Acknowledgments

      With special thanks to the Dunblane and Shotts abattoirs for donating sheep heart for use in this study. We also thank Benedict Reilly O'Donnell and Gavin Robertson for providing technical assistance with measurements of free Zn2+ and assisting with the single channel TPEN data. We are grateful to Dr. Walter Geibert (University of Edinburgh, Grant Institute) for technical assistance with the ICP-OES analysis. We also thank Prof. Richard Thompson (University of Maryland) for advice on performing accurate measurements of free zinc at very low concentrations.

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