Article

Protein Phosphatases Decrease Sarcoplasmic Reticulum Calcium Content by Stimulating Calcium Release in Cardiac Myocytes

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Abstract

Phosphorylation/dephosphorylation of Ca2+ transport proteins by cellular kinases and phosphatases plays an important role in regulation of cardiac excitation-contraction coupling; furthermore abnormal protein kinase and phosphatase activities have been implicated in heart failure. However, the precise mechanisms of action of these enzymes on intracellular Ca2+ handling in normal and diseased hearts remains poorly understood. We have investigated the effects of protein phosphatases PP1 and PP2A on spontaneous Ca2+ sparks and SR Ca2+ load in myocytes permeabilized with saponin. Exposure of myocytes to PP1 or PP2A caused a dramatic increase in frequency of Ca2+ sparks followed by a nearly complete disappearance of events. These effects were accompanied by depletion of the SR Ca2+ stores, as determined by application of caffeine. These changes in Ca2+ release and SR Ca2+ load could be prevented by the inhibitors of PP1 and PP2A phosphatase activities okadaic acid and calyculin A. At the single channel level, PP1 increased the open probability of RyRs incorporated into lipid bilayers. PP1-mediated RyR dephosphorylation in our permeabilized myocytes preparations was confirmed biochemically by quantitative immunoblotting using a phosphospecific anti-RyR antibody. Our results suggest that increased intracellular phosphatase activity stimulates RyR-mediated SR Ca2+ release leading to depleted SR Ca2+ stores in cardiac myocytes.

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... Overall, in the majority of studies, phosphatases reportedly downregulate SR Ca 2+ release and contractile performance (Neumann et al., 1993; duBell et al., 1996, 2002 ; Carr et al., 2002; Santana et al., 2002). Application of PP1 and PP2A to permeabilized myocytes caused an acute activation of Ca 2+ sparks followed by depletion of the SR Ca 2+ content (Terentyev et al., 2003a), suggesting that depression of EC coupling by phosphatases may involve depletion of the SR Ca 2+ stores due to increased activation of RyR2s. Consistent with this possibility, phosphatases have been shown to increase RyR2 activity in some (Lokuta et al., 1995; Terentyev et al., 2003a; Carter et al., 2006) but not all single channel studies (Hain et al., 1994). ...
... Application of PP1 and PP2A to permeabilized myocytes caused an acute activation of Ca 2+ sparks followed by depletion of the SR Ca 2+ content (Terentyev et al., 2003a), suggesting that depression of EC coupling by phosphatases may involve depletion of the SR Ca 2+ stores due to increased activation of RyR2s. Consistent with this possibility, phosphatases have been shown to increase RyR2 activity in some (Lokuta et al., 1995; Terentyev et al., 2003a; Carter et al., 2006) but not all single channel studies (Hain et al., 1994). Overall, as summarized in this brief overview, the mechanisms and functional consequences of RyR2 phosphorylation remain to be conclusively elucidated. ...
... For example, buffering [Ca 2+ ]SR using low affinity exogenous Ca 2+ buffers such as citrate or maleate prolonged the duration of Ca 2+ release during both Ca 2+ sparks and global Ca 2+ transients (Terentyev et al., 2002). Subsequently, similar results were obtained by varying the expression level (increasing or decreasing) of the endogenous Ca 2+ buffer CASQ2 in cardiac myocytes (Terentyev et al., 2003b). Recently, the role of this mechanism has received further support from the observation that Ca 2+ sparks terminate at a constant free [Ca 2+ ] SR regardless of the extent of [Ca 2+ ] SR buffering by CASQ2 (Terentyev et al., 2008). ...
Article
In the heart, Ca(2+) released from the intracellular Ca(2+) storage site, the sarcoplasmic reticulum (SR), is the principal determinant of cardiac contractility. SR Ca(2+) release is controlled by dedicated molecular machinery, composed of the cardiac ryanodine receptor (RyR2) and a number of accessory proteins, including FKBP12.6, calsequestrin (CASQ2), triadin (TRD) and junctin (JN). Acquired and genetic defects in the components of the release channel complex result in a spectrum of abnormal Ca(2+) release phenotypes ranging from arrhythmogenic spontaneous Ca(2+) releases and Ca(2+) alternans to the uniformly diminished systolic Ca(2+) release characteristic of heart failure. In this article, we will present an overview of the structure and molecular components of the SR and Ca(2+) release machinery and its modulation by different intracellular factors, such as Ca(2+) levels inside the SR as well as phosphorylation and redox modification of RyR2s. We will also discuss the relationships between abnormal SR Ca(2+) release and various cardiac disease phenotypes, including, arrhythmias and heart failure, and consider SR Ca(2+) release as a potential therapeutic target.
... Indeed, a number of studies reported no discernible effects of PKA stimulation on RyR2 Ca 2+ release (Li et al. 2002;Stange et al. 2003;Benkusky et al. 2007). Interestingly, previous work by this group and others has shown that decreasing (rather than increasing) RyR2 phosphorylation at this site by exogenous phosphatases results in increased RyR2 activity and enhanced diastolic SR Ca 2+ release (Lokuta et al. 1995;Valdivia et al. 1995;Terentyev et al. 2003). These controversial results further suggest that the role of RyR2 Ser-2808 phosphorylation is more complex than currently recognized. ...
... Marks and coworkers have provided evidence in support of such a role via phosphorylation on Ser-2808 increasing RyR2 activity (Marx et al. 2000;Wehrens et al. 2006;Shan et al. 2010). Yet others found no indications that RyR2 PKA phosphorylation perceptibly affected Ca 2+ handling and contractility in normal and diseased hearts (Li et al. 2002;Xiao et al. 2005;Benkusky et al. 2007;Respress et al. 2012;Zhang et al. 2012); some studies even reported that RyR2 dephosphorylation (rather than phosphorylation) at this site increased RyR2 activity Terentyev et al. 2003). This long lasting controversy is a likely indication that RyR2 regulation is more complex than currently realized, thus resulting in a range of apparently inconsistent results. ...
... Moreover, [ 3 H]ryanodine binding was significantly increased in TM hearts, especially in the TM-S2808A+/− group, which is indicative of increased RyR2 activity in these groups (Fig. 7). These results are also supported by previous reports showing that RyR2 dephosphorylation can increase RyR2 activity in various experimental settings (Lokuta et al. 1995;Valdivia et al. 1995;Terentyev et al. 2003). The heterozygous-disadvantaged manner in which genetic modification affected the RyR2 tetramer is not unique to this channel. ...
Article
Phosphorylation of the cardiac ryanodine receptor (RyR2) by protein kinase A (PKA) at Ser-2808 is suggested to mediate the physiological "fight or flight" response and contribute to heart failure by rendering the sarcoplasmic reticulum (SR) leaky for Ca2+. In the present study, we examined the potential role of RyR2 phosphorylation at Ser-2808 in the progression of Ca2+-dependent cardiomyopathy (CCM) by using mice genetically modified to feature elevated SR Ca2+ leak while expressing RyR2s that cannot be phosphorylated at this site (S2808A). Surprisingly, rather than alleviating the disease phenotype, constitutive dephosphorylation of Ser-2808 aggravated CCM as manifested by shortened survival, deteriorated in vivo cardiac function, exacerbated SR Ca2+ leak and mitochondrial injury. Notably, the deteriorations of cardiac function, myocyte Ca2+ handling, and mitochondria integrity were consistently worse in mice with heterozygous ablation of Ser-2808 than in mice with complete ablation. Wild type (WT) and CCM myocytes expressing unmutated RyR2s exhibited a high level of baseline phosphorylation at Ser-2808. Exposure of these CCM cells to protein phosphatase 1 caused a transitory increase in Ca2+ leak attributable to partial dephosphorylation of RyR2 tetramers at Ser-2808 from more fully phosphorylated state. Thus, exacerbated Ca2+ leak through partially dephosphorylated RyR2s accounts for the prevalence of the disease phenotype in the heterozygous S2808A CCM mice. These results do not support the importance of RyR2 hyperphosphorylation in Ca2+-dependent heart disease, and rather suggest roles for the opposite process, the RyR2 dephosphorylation at this residue in physiological and pathophysiological Ca2+ signaling.
... Ca 2+ spark size was also significantly increased in HF. Width of Ca 2+ sparks was increased by 37 ± 3% (2.82 ± 0.08 vs. 2.06 ± 0.14 μm, P < 0.01; Figure 2D) and duration was increased by 44 ± 4% (39.54 ± 1.57 vs. 27.39 ± 2.41 ms, P < 0.05; Figure 2E). ...
... These data contradict results derived from permeabilized rat CMs showing increased diastolic Ca 2+ loss from the SR upon exposure to PP1 and PP2A, which was accompanied by a depletion of SR Ca 2+ stores. 27 However, the experimental set-ups were different: while PDP3 disrupts the interactions of PP1 with regulatory subunits thereby releasing free catalytically active endogenous PP1 catalytic subunit that can dephosphorylate nearby substrates, adding recombinant PP1 and PP2A will have different effects regarding their concentration in cellular compartments, the posttranslational modifications on them and the metal content depending on the source of the recombinant proteins, 28 which can result in different phenotypes. ...
Article
Background Disruption of Ca²⁺ homeostasis is a key pathomechanism in heart failure. CaMKII‐dependent hyperphosphorylation of ryanodine receptors in the sarcoplasmic reticulum (SR) increases the arrhythmogenic SR Ca²⁺ leak and depletes SR Ca²⁺ stores. The contribution of conversely acting serine/threonine phosphatases [protein phosphatase 1 (PP1) and 2A (PP2A)] is largely unknown. Methods and results Human myocardium from three groups of patients was investigated: (i) healthy controls (non‐failing, NF, n = 8), (ii) compensated hypertrophy (Hy, n = 16), and (iii) end‐stage heart failure (HF, n = 52). Expression of PP1 was unchanged in Hy but greater in HF compared to NF while its endogenous inhibitor‐1 (I‐1) was markedly lower expressed in both compared to NF, suggesting increased total PP1 activity. In contrast, PP2A expression was lower in Hy and HF compared to NF. Ca²⁺ homeostasis was severely disturbed in HF compared to Hy signified by a higher SR Ca²⁺ leak, lower systolic Ca²⁺ transients as well as a decreased SR Ca²⁺ load. Inhibition of PP1/PP2A by okadaic acid increased SR Ca²⁺ load and systolic Ca²⁺ transients but severely aggravated diastolic SR Ca²⁺ leak and cellular arrhythmias in Hy. Conversely, selective activation of PP1 by a PP1‐disrupting peptide (PDP3) in HF potently reduced SR Ca²⁺ leak as well as cellular arrhythmias and, importantly, did not compromise systolic Ca²⁺ release and SR Ca²⁺ load. Conclusion This study is the first to functionally investigate the role of PP1/PP2A for Ca²⁺ homeostasis in diseased human myocardium. Our data indicate that a modulation of phosphatase activity potently impacts Ca²⁺ cycling properties. An activation of PP1 counteracts increased kinase activity in heart failure and successfully seals the arrhythmogenic SR Ca²⁺ leak. It may thus represent a promising future antiarrhythmic therapeutic approach.
... Activité des canaux à ions dans les muscles cardiaques et squelettiques , Terentyev D. 2003 ...
... Récepteur à la ryanodine 2 Activité des canaux à ions dans les muscles cardiaques (Terentyev D, 2003, (Marx SO. 2000)) SFPQ PSF Epissage (Hirano K. 1996 ...
Article
Le paludisme (ou malaria) est la parasitose la plus répandue à travers le monde (WHO, 2011).La moitié de la population mondiale est exposée à cette maladie causée par le protozoairePlasmodium. La résistance aux traitements qui se développe chez le parasite représente un véritableobstacle à la mise en place de programmes de lutte globale. Le développement de nouveauxprotocoles thérapeutiques plus efficaces ciblant les apicomplexes Plasmodium passe par uneamélioration de nos connaissances concernant la biologie fondamentale des parasites. Par ce biais,nous pourrons identifier de nouvelles cibles originales visant des mécanismes essentiels etspécifiques au développement du pathogène. Chez Plasmodium falciparum (l'espèce responsable dela forme la plus mortelle de malaria), la phase érythrocytaire qui se déroule chez son hôte humain estrelativement courte (48 heures). Le parasite y subit un grand nombre de changementsmorphologiques nécessitant une différentiation précise, spécifique et régulée au cours du temps.Parmi les éléments pouvant contrôler ces mécanismes, les phénomènes de phosphorylationréversibles semblent être des candidats de choix.Nous avons, dans un premier temps, entrepris la caractérisation de PfI2. Ce travail a permisd'identifier cette protéine comme étant un régulateur négatif de PfPP1 localisé au niveaunucléocytoplasmique et indispensable au développement érythrocytaire du parasite. Des étudesd'interaction in vitro et in vivo ont permis d'identifier un certain nombre de résidus impliqués dans lafixation et la fonction de PfI2. En nous basant sur ces résultats, nous avons entrepris une explorationplus précise des relations structure/fonction du complexe PfI2/PfPP1.Concernant l'identification de PfI3, nous avons récemment publiée dans le Journal ofBiological Chemistry (Frèville A. 2012) un travail montrant que chez le parasite, l'inhibiteur 3 estlocalisé au niveau nucléaire et est essentiel au développement asexuel du pathogène. Desexpériences d'interaction in vitro ont permis de montrer que PfI3 est capable de se fixer in vitro àPfPP1 et ce principalement via le motif primaire RVxF. Chez des levures déplétées de leur inhibiteur3, l'expression épisomique de PfI3 n'a pas permis de restaurer la croissance des cellules. Desexpériences in vitro d'activités phosphatase révèlent une action positive de PfI3 sur PfPP1. Cerésultat, inverse de celui que l'on observe chez ses homologues chez la levure ou l'humain met enévidence une fonction différente et spécifique de PfI3 et font de cette protéine un régulateurnucléaire potentiel de PfPP1 chez Plasmodium falciparum.L'ensemble de ce travail de thèse à permis d'identifier et de caractériser chez Plasmodiumfalciparum deux régulateurs potentiels de la phosphatase de type 1 mais également de mettre enévidence un certain nombre d'éléments spécifiques au fonctionnement et au développement duparasite faisant de ces protéines des cibles thérapeutiques intéressantes.
... Importantly, we showed that dephosphorylation of RyR2 incorporated into lipid bilayers with PP1 also increases RyR2 Po [66]. Carter et al. [59,61] demonstrated that PP1-mediated increase in RyR2 activity is not associated with changes in sensitivity to cytosolic Ca 2+ and stems from abbreviation of closed states of the channel. ...
... Carter et al. [59,61] demonstrated that PP1-mediated increase in RyR2 activity is not associated with changes in sensitivity to cytosolic Ca 2+ and stems from abbreviation of closed states of the channel. At the cellular level, exposure of rat ventricular myocytes with plasmamembrane permeabilized with saponin to catalytic subunits of PP1 and PP2A resulted in dramatic increase in RyR2-mediated SR Ca 2+ leak and rapid depletion of SR Ca 2+ stores, preventable by preincubation of myocytes with phosphatase inhibitors [66]. To prevent potential effects of depletion of the stores which confound interpretation of results, Liu et al. [67] used mouse model overexpressing skeletal type of SR Ca 2+ ATPase SERCa1a. ...
Article
The amount and timing of Ca(2+) release from the sarcoplasmic reticulum (SR) during cardiac cycle are the main determinants of cardiac contractility. Reversible phosphorylation of the SR Ca(2+) release channel, ryanodine receptor type 2 (RyR2) is the central mechanism of regulation of Ca(2+) release in cardiomyocytes. Three major serine-threonine phosphatases including PP1, PP2A and PP2B (calcineurin) have been implicated in modulation of RyR2 function. Changes in expression levels of these phosphatases, their activity and targeting to the RyR2 macromolecular complex were demonstrated in many animal models of cardiac disease and humans and are implicated in cardiac arrhythmia and heart failure. Here we review evidence in support of regulation of RyR2-mediated SR Ca(2+) release by serine-threonine phosphatases and the role and mechanisms of dysregulation of phosphatases in various disease states.
... Activité des canaux à ions dans les muscles cardiaques et squelettiques , Terentyev D. 2003 ...
... Récepteur à la ryanodine 2 Activité des canaux à ions dans les muscles cardiaques (Terentyev D, 2003, (Marx SO. 2000)) SFPQ PSF Epissage (Hirano K. 1996 ...
Thesis
Full-text available
Le paludisme (ou malaria) est la parasitose la plus répandue à travers le monde (WHO, 2011). La moitié de la population mondiale est exposée à cette maladie causée par le protozoaire Plasmodium. La résistance aux traitements qui se développe chez le parasite représente un véritable obstacle à la mise en place de programmes de lutte globale. Le développement de nouveaux protocoles thérapeutiques plus efficaces ciblant les apicomplexes Plasmodium passe par une amélioration de nos connaissances concernant la biologie fondamentale des parasites. Par ce biais,nous pourrons identifier de nouvelles cibles originales visant des mécanismes essentiels et spécifiques au développement du pathogène. Chez Plasmodium falciparum (l'espèce responsable de la forme la plus mortelle de malaria), la phase érythrocytaire qui se déroule chez son hôte humain est relativement courte (48 heures). Le parasite y subit un grand nombre de changements morphologiques nécessitant une différentiation précise, spécifique et régulée au cours du temps.Parmi les éléments pouvant contrôler ces mécanismes, les phénomènes de phosphorylation réversibles semblent être des candidats de choix.Nous avons, dans un premier temps, entrepris la caractérisation de PfI2. Ce travail a permis d'identifier cette protéine comme étant un régulateur négatif de PfPP1 localisé au niveau nucléocytoplasmique et indispensable au développement érythrocytaire du parasite. Des études d'interaction in vitro et in vivo ont permis d'identifier un certain nombre de résidus impliqués dans la fixation et la fonction de PfI2. En nous basant sur ces résultats, nous avons entrepris une exploration plus précise des relations structure/fonction du complexe PfI2/PfPP1.Concernant l'identification de PfI3, nous avons récemment publiée dans le Journal of Biological Chemistry (Frèville A. 2012) un travail montrant que chez le parasite, l'inhibiteur 3 est localisé au niveau nucléaire et est essentiel au développement asexuel du pathogène. Des expériences d'interaction in vitro ont permis de montrer que PfI3 est capable de se fixer in vitro àPfPP1 et ce principalement via le motif primaire RVxF. Chez des levures déplétées de leur inhibiteur 3, l'expression épisomique de PfI3 n'a pas permis de restaurer la croissance des cellules. Des expériences in vitro d'activités phosphatase révèlent une action positive de PfI3 sur PfPP1. Ce résultat, inverse de celui que l'on observe chez ses homologues chez la levure ou l'humain met en évidence une fonction différente et spécifique de PfI3 et font de cette protéine un régulateur nucléaire potentiel de PfPP1 chez Plasmodium falciparum. L'ensemble de ce travail de thèse à permis d'identifier et de caractériser chez Plasmodium falciparum deux régulateurs potentiels de la phosphatase de type 1 mais également de mettre en évidence un certain nombre d'éléments spécifiques au fonctionnement et au développement du parasite faisant de ces protéines des cibles thérapeutiques intéressantes.
... Phosphorylation at S2808, S2814 and S2031 to maximal levels is associated with increased RyR2 Po and subsequent treatment with PP1 reduces this activity in several studies (Carter et al. 2006;Carter et al. 2011). Interestingly in single channel experiments, dephosphorylation of RyR2 at S2808 by PP1 also increases Po (Carter et al. 2006;Terentyev et al. 2003). Carter et al. have shown that this activity is not associated with changes in sensitivity to cytosolic Ca 2+ , instead stemming from shortening of the closed states of the channel (Carter et al. 2011;Carter et al. 2006). ...
... Györke and colleagues utilized their mouse model of Ca 2+ -dependent cardiomyopathy (CCM), with CSQ2 KO and SERCa1a overexpression, to examine the potential role of S2808 phosphorylation in the development of the disease (Ho et al. 2014;Kalyanasundaram 2013 resulted in a dramatic increase in leak and a rapid depletion of SR Ca 2+ stores, suggestive that this leak is due to the change in RyR2 function when dephosphorylated (Terentyev et al. 2003). These data also implicate dephosphorylation in increasing RyR2 channel activity (Marx et al. 2000). ...
... The situation is further complicated because completely desphosphorylating RyR2 using protein phosphatase 1 (PP1; a phosphatase known to associate with RyR2) from basal levels where the channel is 75 % phosphorylated at S2808, actually causes an increase in Po [6]. Although the basal levels of S2808 phosphorylation were not calculated for singlechannel experiments, Terentyev et al. (2003) also showed that PP1 increased the Po of RyR2 and that both PP1 and protein phosphatase 2 (PP2A) increased Ca 2 + -induced Ca 2 + -wave frequency and decreased SR Ca 2 + content in permeabilized cardiomyocytes [11]. Figure 2 shows how Po is altered in parallel with S2808 phosphorylation levels. ...
... The situation is further complicated because completely desphosphorylating RyR2 using protein phosphatase 1 (PP1; a phosphatase known to associate with RyR2) from basal levels where the channel is 75 % phosphorylated at S2808, actually causes an increase in Po [6]. Although the basal levels of S2808 phosphorylation were not calculated for singlechannel experiments, Terentyev et al. (2003) also showed that PP1 increased the Po of RyR2 and that both PP1 and protein phosphatase 2 (PP2A) increased Ca 2 + -induced Ca 2 + -wave frequency and decreased SR Ca 2 + content in permeabilized cardiomyocytes [11]. Figure 2 shows how Po is altered in parallel with S2808 phosphorylation levels. ...
Article
Once opened, ryanodine receptors (RyR) are efficient pathways for the release of Ca2+ from the endoplasmic/sarcoplasmic reticulum (ER/SR). The precise nature of the Ca2+-release event, however, requires fine-tuning for the specific process and type of cell involved. For example, the spatial organization of RyRs, the luminal [Ca2+] and the influence of soluble regulators that fluctuate under physiological and pathophysiological control mechanisms, all affect the amplitude and duration of RyR Ca2+ fluxes. Various proteins are docked tightly to the huge bulky structure of RyR and there is growing evidence that, together, they provide a sophisticated and integrated system for regulating RyR channel gating. This review focuses on those proteins that are relevant to phosphorylation of RyR channels with particular reference to the cardiac isoform of RyR (RyR2). How phosphorylation of RyR affects channel activity and whether proteins such as the FK-506 binding proteins (FKBP12 and FKBP12.6) are involved, have been highly controversial subjects for more than a decade. But that is expected given the large number of participating proteins, the relevance of phosphorylation in heart failure and inherited arrhythmic diseases, and the frustrations of predicting relationships between structure and function before the advent of a high resolution structure of RyR.
... PP2A is often responsible for removal of phosphate groups at PKA phosphorylation sites in cardiac muscle and dephosphorylates cTnI (MacDougall et al., 1991), myosin-binding protein C ), phospholamban (MacDougall et al., 1991, inwardly rectifier potassium channels (Barros et al., 1993) and Ltype Ca 2+ channels (Davare et al., 2000) (Table 1). PP2A has a stimulatory effect on generation of Ca 2+ sparks (Terentyev et al., 2003) and regulates transcription factors such as CREB (Wadzinski et al., 1993) and NFκB (Yang et al., 2001), which are involved in cardiac remodeling (Fentzke et al., 1998;Frantz et al., 2003). These findings indicate that PP2A may function to balance the effects of β-adrenergic stimulation in the heart. ...
... It is a paradox that phosphorylation of RyR by PKA has no effect on generation of Ca 2+ sparks (Sobie et al., 2006). On the other hand, PP2A appears to increase the frequency of Ca 2+ sparks transiently (Terentyev et al., 2003). Sheehan et al., recently reported an inhibition of spark frequency in heart cells expressing caPak1 (Sheehan et al., 2009). ...
Article
Cardiac excitation and contraction are regulated by a variety of signaling molecules. Central to the regulatory scheme are protein kinases and phosphatases that carry out reversible phosphorylation of different effectors. The process of beta-adrenergic stimulation mediated by cAMP dependent protein kinase (PKA) forms a well-known pathway considered as the most significant control mechanism in excitation and contraction as well as many other regulatory mechanisms in cardiac function. However, although dephosphorylation pathways are critical to these regulatory processes, signaling to phosphatases is relatively poorly understood. Emerging evidence indicates that regulation of phosphatases, which dampen the effect of beta-adrenergic stimulation, is also important. We review here functional studies of p21 activated kinase-1 (Pak1) and its potential role as an upstream signal for protein phosphatase PP2A in the heart. Pak1 is a serine/threonine protein kinase directly activated by the small GTPases Cdc42 and Rac1. Pak1 is highly expressed in different regions of the heart and modulates the activities of ion channels, sarcomeric proteins, and other phosphoproteins through up-regulation of PP2A activity. Coordination of Pak1 and PP2A activities is not only potentially involved in regulation of normal cardiac function, but is likely to be important in patho-physiological conditions.
... On the other hand, other groups have suggested that PKA phosphorylation of RyR2 is so essential to intracellular Ca 2+ homeostasis that derangement of this process may be the basis for heart failure (HF) [37,90] and catecholaminergic polymorphic ventricular tachycardia (CPVT) episodes [91]. Between these two extremes, other results, mainly from in vitro experiments, imply that PKA phosphorylation increases [86,92], decreases [74,93] or has no effect [94] on RyR2 activity. Several factors preclude an easy interpretation of phosphorylation results. ...
Article
Full-text available
Excitation-contraction coupling involves the faithful conversion of electrical stimuli to mechanical shortening in striated muscle cells, enabled by the ubiquitous second messenger, calcium. Crucial to this process are ryanodine receptors (RyRs), the sentinels of massive intracellular calcium stores contained within the sarcoplasmic reticulum. In response to sarcolemmal depolarization, RyRs release calcium into the cytosol, facilitating mobilization of the myofilaments and enabling cell contraction. In order for the cells to relax, calcium must be rapidly resequestered or extruded from the cytosol. The sustainability of this cycle is crucially dependent upon precise regulation of RyRs by numerous cytosolic metabolites and by proteins within the lumen of the sarcoplasmic reticulum and those directly associated with the receptors in a macromolecular complex. In addition to providing the majority of the calcium necessary for contraction of cardiac and skeletal muscle, RyRs act as molecular switchboards that integrate a multitude of cytosolic signals such as dynamic and steady calcium fluctuations, β-adrenergic stimulation (phosphorylation), nitrosylation and metabolic states, and transduce these signals to the channel pore to release appropriate amounts of calcium. Indeed, dysregulation of calcium release via RyRs is associated with life-threatening diseases in both skeletal and cardiac muscle. In this paper, we briefly review some of the most outstanding structural and functional attributes of RyRs and their mechanism of regulation. Further, we address pathogenic RyR dysfunction implicated in cardiovascular disease and skeletal myopathies.
... It has been reported that both PKA-dependent and CaMKII-dependent phosphorylation of RyR2 can give rise to an increase in open probability (P o ) (Marx et al. 2000; Carter et al. 2006; Wehrens et al. 2004). On the other hand, we, and others, have shown that dephosphorylating RyR2 can also increase P o , indicating that there must be an inhibitory component to phosphorylation (Carter et al. 2006; Terentyev et al. 2003; Lokuta et al. 1995). The aim of this study was, therefore, to examine the underlying changes in RyR2 single-channel gating that occur following phosphorylation of RyR2. ...
Article
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Phosphorylation of the cardiac ryanodine receptor (RyR2) is thought to be important not only for normal cardiac excitation-contraction coupling but also in exacerbating abnormalities in Ca2+ homeostasis in heart failure. Linking phosphorylation to specific changes in the single-channel function of RyR2 has proved very difficult, yielding much controversy within the field. We therefore investigated the mechanistic changes that take place at the single-channel level after phosphorylating RyR2 and, in particular, the idea that PKA-dependent phosphorylation increases RyR2 sensitivity to cytosolic Ca2+. We show that hyperphosphorylation by exogenous PKA increases open probability (P o) but, crucially, RyR2 becomes uncoupled from the influence of cytosolic Ca2+; lowering [Ca2+] to subactivating levels no longer closes the channels. Phosphatase (PP1) treatment reverses these gating changes, returning the channels to a Ca2+-sensitive mode of gating. We additionally found that cytosolic incubation with Mg2+/ATP in the absence of exogenously added kinase could phosphorylate RyR2 in approximately 50% of channels, thereby indicating that an endogenous kinase incorporates into the bilayer together with RyR2. Channels activated by the endogenous kinase exhibited identical changes in gating behavior to those activated by exogenous PKA, including uncoupling from the influence of cytosolic Ca2+. We show that the endogenous kinase is both Ca2+-dependent and sensitive to inhibitors of PKC. Moreover, the Ca2+-dependent, endogenous kinase–induced changes in RyR2 gating do not appear to be related to phosphorylation of serine-2809. Further work is required to investigate the identity and physiological role of this Ca2+-dependent endogenous kinase that can uncouple RyR2 gating from direct cytosolic Ca2+ regulation.
... Thus changes in cytosolic Ca 2+ may regulate RyR directly but may also regulate RyR indirectly via the actions of the Ca 2+sensitive proteins CaM and CAMKII. RyR channels can be phosphorylated at multiple sites by a range of kinases [64][65][66][67][68] causing marked changes to channel activity including both increases and decreases in Po [45,[69][70][71]. RyR phosphorylation is therefore an extremely flexible, yet complex system by which intracellular Ca 2+-release can be regulated. ...
Article
It was first proposed that cyclic ADP-ribose (cADPR) could activate ryanodine receptors (RyR) in 1991. Following a subsequent report that cADPR could activate cardiac RyR (RyR2) reconstituted into artificial membranes and stimulate Ca(2+) -release from isolated cardiac SR, there has been a steadily mounting stockpile of publications proclaiming the physiological and pathophysiological importance of cADPR in the cardiovascular system. It was only 2 years earlier, in 1989, that cADPR was first identified as the active metabolite of nicotinamide adenine dinucleotide (NAD), responsible for triggering the release of Ca(2+) from crude homogenates of sea urchin eggs. Twenty years later, can we boast of being any closer to unraveling the mechanisms by which cADPR modulates intracellular Ca(2+) -release? This review sets out to examine the mechanisms underlying the effects of cADPR and ask whether cADPR is an important signaling molecule in the heart.
... Within a murine model of asthma, ASMC local Ca 2ϩ signaling is increased (41); it is unknown how this increase is maintained, and it might seem reasonable to suggest that the canonical transient receptor potential-3 channel, which mediates extracellular Ca 2ϩ influx and has increased expression and activity in asthma (45), may play a role in maintaining this signaling. We have further reinforced the specific role for the protein phosphatase CaN by finding out that inhibition of PP2A with endothall does not affect local Ca 2ϩ signals (Fig. 1E), unlike in cardiac myocytes where PP2A was shown to increase Ca 2ϩ sparks (40). RyRs are essential for spark formation; however, IP 3 Rs cross talk with RyRs, promoting Ca 2ϩ spark formation through a local CICR process (25,26). ...
... This experiment also shows that the contribution of RyR cannot be judged without controlling for SR Ca 2+ load. Others have studied isolated RyR2 preparations and observed that not only phosphorylation at S2808, but also dephosphorylation, increased its open probability (9,11). Several groups have failed to reproduce the effect observed by Marks and colleagues (2) of PKA phosphorylation at S2808 or mutations of this site on FKBP12.6 binding (2, 10, 12, 13), but others have (14). ...
Article
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In situations of stress the heart beats faster and stronger. According to Marks and colleagues, this response is, to a large extent, the consequence of facilitated Ca²+ release from intracellular Ca²+ stores via ryanodine receptor 2 (RyR2), thought to be due to catecholamine-induced increases in RyR2 phosphorylation at serine 2808 (S2808). If catecholamine stimulation is sustained (for example, as occurs in heart failure), RyR2 becomes hyperphosphorylated and "leaky," leading to arrhythmias and other pathology. This "leaky RyR2 hypothesis" is highly controversial. In this issue of the JCI, Marks and colleagues report on two new mouse lines with mutations in S2808 that provide strong evidence supporting their theory. Moreover, the experiments revealed an influence of redox modifications of RyR2 that may account for some discrepancies in the field.
... Thus, high levels of peroxynitrite shift the kinase/phosphatase balance leading to decreased PLB phosphorylation. While PP2A also modulates RyR phosphorylation (Terentyev et al., 2003), we did not observe any effects of high peroxynitrite on the β-AR response in PLB knockout myocytes, which suggests that this may be a compartmentalized effect targeted to PLB. ...
Article
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Peroxynitrite is a potent oxidant that is quickly emerging as a crucial modulator of myocardial function. This review will focus on the regulation of myocardial contraction by peroxynitrite during health and disease, with a specific emphasis on cardiomyocyte Ca2+ handling, proposed signaling pathways, and protein end-targets. The heart plays a vital role in the cardiovascular system by providing the specialized tissues and organs of the body with a continual supply of oxygen and other essential nutrients. Modulation of myocardial contraction allows the heart to meet the demands of the body despite continual changes in metabolism. This modulation occurs at several different levels, including the level of the ventricular cardiomyocyte.
... Some prior work has shown that Ser2808 can be hyperphosphorylated by PKA (45) and that PKA-dependent phosphorylation of RyR2 can increase the open probability of RyR2 (45)(46)(47). In contrast, work by others has shown that dephosphorylation of RyR2 can also increase open probability, indicating that there must be an inhibitory component to phosphorylation (46,48,49). The controversy about the function of RyR2 phosphorylation in the myocardium is highlighted by recent work from Houser's group (50), in which genetically manipulated mice with a RyR2 S2808A mutation were produced that could not undergo Integrins modulated RyR2 Ser2808 phosphorylation. ...
Article
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Ischemic damage is recognized to cause cardiomyocyte (CM) death and myocardial dysfunction, but the role of cell-matrix interactions and integrins in this process has not been extensively studied. Expression of α7β1D integrin, the dominant integrin in normal adult CMs, increases during ischemia/reperfusion (I/R), while deficiency of β1 integrins increases ischemic damage. We hypothesized that the forced overexpression of integrins on the CM would offer protection from I/R injury. Tg mice with CM-specific overexpression of integrin α7β1D exposed to I/R had a substantial reduction in infarct size compared with that of α5β1D-overexpressing mice and WT littermate controls. Using isolated CMs, we found that α7β1D preserved mitochondrial membrane potential during hypoxia/reoxygenation (H/R) injury via inhibition of mitochondrial Ca2+ overload but did not alter H/R effects on oxidative stress. Therefore, we assessed Ca2+ handling proteins in the CM and found that β1D integrin colocalized with ryanodine receptor 2 (RyR2) in CM T-tubules, complexed with RyR2 in human and rat heart, and specifically bound to RyR2 amino acids 165-175. Integrins stabilized the RyR2 interdomain interaction, and this stabilization required integrin receptor binding to its ECM ligand. These data suggest that α7β1D integrin modifies Ca2+ regulatory pathways and offers a means to protect the myocardium from ischemic injury.
... The myocytes were divided for molecular, biochemical, and electrophysiological assays and stored at room temperature until use. Isolation of rat ventricular myocytes was performed as previously described [45]. Isolated myocytes were plated on laminin-coated glass coverslips in serumfree medium 199 supplemented with (in mM): 25 NaHCO 3 , 5 creatine, 5 taurine, 10 U/ml penicillin, 10 mg/ml streptomycin, and 10 mg/ml gentamycin (pH 7.3). ...
... However, it is also possible for peroxynitrite and PP2a to target other proteins within the cardiomyocyte. Additional targets for PP2a include the L-type Ca 2+ channel, troponin I, and the ryanodine receptor [27,28]. We previously demonstrated that NOS2 expression depresses ryanodine receptor function through a cGMP-independent signaling pathway, perhaps via peroxynitrite formation [29]. ...
Article
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High levels of peroxynitrite have been shown to decrease cardiomyocyte contraction through a reduction in phospholamban (PLB) phosphorylation. However, previous reports did not examine the direct effect of peroxynitrite on protein phosphatase activity in the myocardium or the role of specific phosphatases. Here we test the effect of the peroxynitrite donor SIN-1 on protein phosphatase activity in whole heart homogenates, as well as the interaction of PLB with protein phosphatase 1 (PP1) and 2a (PP2a). SIN-1 (200 μmol/L) induced a significant increase in protein phosphatase activity, which was alleviated with the specific PP1/PP2a inhibitor okadaic acid. Conversely, lower concentrations of SIN-1 and the nitric oxide donor spermine NONOate (300 μmol/L) were both without effect on phosphatase activity. We next examined the effect of SIN-1 on the interaction of PLB with PP1 and PP2a using co-immunoprecipitation, since okadaic acid inhibited the effects of SIN-1 in our current and previous studies. SIN-1 significantly increased the interaction of PLB with PP2a, but had no effect on the interaction between PLB and PP1. Urate, a peroxynitrite scavenger, inhibited the effects of SIN-1 on phosphatase activity and the interaction of PLB with PP2a, thus implicating peroxynitrite as the causal species. The results of this study provide further insight into the mechanism through which high levels of peroxynitrite serve to decrease PLB phosphorylation and myocardial contraction. Therefore, peroxynitrite signaling could play a key role in the contractile dysfunction manifested in heart failure where peroxynitrite production and protein phosphatase activity are increased and PLB phosphorylation is decreased.
... Several studies have demonstrated that PP-mediated dephosphorylation of RyR2 decreased channel open probability, but other studies have reported the opposite. 9,23,61,62 Moreover, it was shown that PP increases RyR2 leakiness in cells expressing wild-type, but not S2808A mutant, RyR2 with the disabled PKA phosphorylation site. 24 Thus, the PP-mediated regulation of RyR2 remains controversial at this time. ...
Article
Cardiac ryanodine receptor type 2 plays a key role in excitation-contraction coupling. The ryanodine receptor type 2 channel protein is modulated by various post-translational modifications, including phosphorylation by protein kinase A and Ca(2+)/calmodulin protein kinase II. Despite extensive research in this area, the functional effects of ryanodine receptor type 2 phosphorylation remain disputed. In particular, the potential involvement of increased ryanodine receptor type 2 phosphorylation in the pathogenesis of heart failure and arrhythmias remains a controversial area, which is discussed in this review article.
... First, as stated above, RyR2 channels contain multiple phosphorylation sites that, depending on their phospho-state, may attenuate or synergize the effect of the other sites, or may require prior phosphorylation to activate the whole protein. This has become evident in experiments in which it has been possible to link variable levels of phosphorylation with defined single-channel activity (Carter et al., 2006) and also where it is clear that phosphatases activate RyR2 channels to higher levels than either PKA or CaMKII alone (Lokuta et al., 1995;Terentyev et al., 2003), suggesting that dephosphorylation uncovers a set of phospho-sites that modulate RyR2 activity but are not affected by either kinase. Second, RyR2 activity has long been established to be dependent on the speed of Ca 2+ application [as inferred by Fabiato in his classical experiments that characterized CICR (Fabiato, 1985) and demonstrated in single channel experiments (Gyorke and Fill, 1993)], and this in turn may greatly influence the overall effect of phosphorylation Jiang et al., 2002). ...
Article
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Ryanodine receptors (RyRs) and inositol triphosphate receptors (InsP3Rs) are structurally related intracellular calcium release channels that participate in multiple primary or secondary amplified Ca(2+) signals, triggering muscle contraction and oscillatory Ca(2+) waves, or activating transcription factors. In the heart, RyRs play an indisputable role in the process of excitation-contraction coupling as the main pathway for Ca(2+) release from sarcoplasmic reticulum (SR), and a less prominent role in the process of excitation-transcription coupling. Conversely, InsP3Rs are believed to contribute in subtle ways, only, to contraction of the heart, and in more important ways to regulation of transcription factors. Because uncontrolled activity of either RyRs or InsP3Rs may elicit life-threatening arrhythmogenic and/or remodeling Ca(2+) signals, regulation of their activity is of paramount importance for normal cardiac function. Due to their structural similarity, many regulatory factors, accessory proteins, and post-translational processes are equivalent for RyRs and InsP3Rs. Here we discuss regulation of RyRs and InsP3Rs by CaMKII phosphorylation, but touch on other kinases whenever appropriate. CaMKII is emerging as a powerful modulator of RyR and InsP3R activity but interestingly, some of the complexities and controversies surrounding phosphorylation of RyRs also apply to InsP3Rs, and a clear-cut effect of CaMKII on either channel eludes investigators for now. Nevertheless, some effects of CaMKII on global cellular activity, such as SR Ca(2+) leak or force-frequency potentiation, appear clear now, and this constrains the limits of the controversies and permits a more tractable approach to elucidate the effects of phosphorylation at the single channel level.
... 25,35 Previously, we reported that exogenous phosphatases, including PP2A, elevate cardiac SR Ca 2ϩ leak by stimulation of ryanodine receptors. 10 This result is in apparent contradiction with the stimulatory effects of increased RyR2 phosphorylation described in the present study. Recently, we found that both phosphorylation and dephosphorylation can stimulate RyR2 activity, resulting in increased SR Ca 2ϩ leak in cardiomyocytes. ...
Article
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MicroRNAs are small endogenous noncoding RNAs that regulate protein expression by hybridization to imprecise complementary sequences of target mRNAs. Changes in abundance of muscle-specific microRNA, miR-1, have been implicated in cardiac disease, including arrhythmia and heart failure. However, the specific molecular targets and cellular mechanisms involved in the action of miR-1 in the heart are only beginning to emerge. In this study we investigated the effects of increased expression of miR-1 on excitation-contraction coupling and Ca(2+) cycling in rat ventricular myocytes using methods of electrophysiology, Ca(2+) imaging and quantitative immunoblotting. Adenoviral-mediated overexpression of miR-1 in myocytes resulted in a marked increase in the amplitude of the inward Ca(2+) current, flattening of Ca(2+) transients voltage dependence, and enhanced frequency of spontaneous Ca(2+) sparks while reducing the sarcoplasmic reticulum Ca(2+) content as compared with control. In the presence of isoproterenol, rhythmically paced, miR-1-overexpressing myocytes exhibited spontaneous arrhythmogenic oscillations of intracellular Ca(2+), events that occurred rarely in control myocytes under the same conditions. The effects of miR-1 were completely reversed by the CaMKII inhibitor KN93. Although phosphorylation of phospholamban was not altered, miR-1 overexpression increased phosphorylation of the ryanodine receptor (RyR2) at S2814 (Ca(2+)/calmodulin-dependent protein kinase) but not at S2808 (protein kinase A). Overexpression of miR-1 was accompanied by a selective decrease in expression of the protein phosphatase PP2A regulatory subunit B56alpha involved in PP2A targeting to specialized subcellular domains. We conclude that miR-1 enhances cardiac excitation-contraction coupling by selectively increasing phosphorylation of the L-type and RyR2 channels via disrupting localization of PP2A activity to these channels.
... The myocytes were divided for molecular, biochemical, and electrophysiological assays and stored at room temperature until use. Isolation of rat ventricular myocytes was performed as previously described [45]. Isolated myocytes were plated on laminin-coated glass coverslips in serumfree medium 199 supplemented with (in mM): 25 NaHCO 3 , 5 creatine, 5 taurine, 10 U/ml penicillin, 10 mg/ml streptomycin, and 10 mg/ml gentamycin (pH 7.3). ...
Article
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In heart failure (HF), arrhythmogenic spontaneous sarcoplasmic reticulum (SR) Ca(2+) release and afterdepolarizations in cardiac myocytes have been linked to abnormally high activity of ryanodine receptors (RyR2s) associated with enhanced phosphorylation of the channel. However, the specific molecular mechanisms underlying RyR2 hyperphosphorylation in HF remain poorly understood. The objective of the current study was to test the hypothesis that the enhanced expression of muscle-specific microRNAs (miRNAs) underlies the HF-related alterations in RyR2 phosphorylation in ventricular myocytes by targeting phosphatase activity localized to the RyR2. We studied hearts isolated from canines with chronic HF exhibiting increased left ventricular (LV) dimensions and decreased LV contractility. qRT-PCR revealed that the levels of miR-1 and miR-133, the most abundant muscle-specific miRNAs, were significantly increased in HF myocytes compared with controls (2- and 1.6-fold, respectively). Western blot analyses demonstrated that expression levels of the protein phosphatase 2A (PP2A) catalytic and regulatory subunits, which are putative targets of miR-133 and miR-1, were decreased in HF cells. PP2A catalytic subunit mRNAs were validated as targets of miR-133 by using luciferase reporter assays. Pharmacological inhibition of phosphatase activity increased the frequency of diastolic Ca(2+) waves and afterdepolarizations in control myocytes. The decreased PP2A activity observed in HF was accompanied by enhanced Ca(2+)/calmodulin-dependent protein kinase (CaMKII)-mediated phosphorylation of RyR2 at sites Ser-2814 and Ser-2030 and increased frequency of diastolic Ca(2+) waves and afterdepolarizations in HF myocytes compared with controls. In HF myocytes, CaMKII inhibitory peptide normalized the frequency of pro-arrhythmic spontaneous diastolic Ca(2+) waves. These findings suggest that altered levels of major muscle-specific miRNAs contribute to abnormal RyR2 function in HF by depressing phosphatase activity localized to the channel, which in turn, leads to the excessive phosphorylation of RyR2s, abnormal Ca(2+) cycling, and increased propensity to arrhythmogenesis.
... Phosphorylation was increased by phosphatase inhibition with 1 μM Calyculin A (Rodriguez et al., 2003;Belevych et al., 2011), or decreased with 20 µM H-89 (Sigma, UK), a PKA inhibitor (Rodriguez et al., 2003;Terentyev et al., 2003;Curran et al., 2007). I decided to focus on 1 μM Calyculin A treatments compared to control as enhanced signal is better for validation purposes, and hyperphosphorylation is an application motivation. ...
... PP1 and PP2A form complexes on ryanodine receptors. In saponin permeabilized myocytes, exposure of PP1 and PP2A dramatically increased Ca sparks with a significant decrease of SR Ca store (Terentyev et al., 2003). On the other hand, targeting of PP2A regulatory subunit B56α by microRNA miR-1 leads to hyperphosphorylation of RyR at the CamKII sites and increases Ca 2+ release and promote cardiac arrhythmogenesis (Terentyev et al., 2009;Belevych et al., 2011). ...
Article
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Calcium transient in cardiomyocytes is regulated by multiple protein kinases and phosphatases. PP2A is a major protein phosphatase in the heart modulating Ca(2+) handling through an array of ion channels, antiporters and pumps, etc. The assembly, localization/translocation, and substrate specificity of PP2A are controlled by different post-translational mechanisms, which in turn are linked to the activities of upstream signaling molecules. Abnormal PP2A expression and activities are associated with defective response to β-adrenergic stimulation and are indication and causal factors in arrhythmia and heart failure.
... CSQ2 is a calcium binding protein that has a high capacity for calcium and is a part of the ryanodine receptor Ca 2+ release complex (Murphy et al., 2011). Additionally, CSQ2 is thought to be involved in providing a readily releasable pool of Ca 2+ to facilitate cardiac contractility as it forms a multi-protein complex with triadin, junctin, and the ryanodine receptor (Scriven et al., 2000) and CSQ2 protein levels appear to be associated with both the level of the SR Ca 2+ store as well as the level of SR Ca 2+ release (Terentyev et al., 2003). Western blot analysis did not reveal any differences in CSQ2 expression between WT and gravin-t/t hearts (Fig. 7E). ...
Article
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Gravin (AKAP12) is an A-kinase-anchoring-protein that scaffolds protein kinase A (PKA), β2-adrenergic receptor (β2-AR), protein phosphatase 2B and protein kinase C. Gravin facilitates β2-AR-dependent signal transduction through PKA to modulate cardiac excitation-contraction coupling and its removal positively affects cardiac contraction. Trabeculae from the right ventricles of gravin mutant (gravin-t/t) mice were employed for force determination. Simultaneously, corresponding intracellular Ca²⁺ transient ([Ca²⁺]i) were measured. Twitch force (Tf)-interval relationship, [Ca²⁺]i-interval relationship, and the rate of decay of post-extrasysolic potentiation (Rf) were also obtained. Western blot analysis were performed to correlate sarcomeric protein expression with alterations in calcium cycling between the WT and gravin-t/t hearts. Gravin-t/t muscles had similar developed force compared to WT muscles despite having lower [Ca²⁺]i at any given external Ca²⁺ concentration ([Ca²⁺]o). The time to peak force and peak [Ca²⁺]i were slower and the time to 75% relaxation was significantly prolonged in gravin-t/t muscles. Both Tf-interval and [Ca²⁺]i-interval relations were depressed in gravin-t/t muscles. Rf, however, did not change. Furthermore, Western blot analysis revealed decreased ryanodine receptor (RyR2) phosphorylation in gravin-t/t hearts. Gravin-t/t cardiac muscle exhibits increased force development in responsiveness to Ca²⁺. The Ca²⁺ cycling across the SR appears to be unaltered in gravin-t/t muscle. Our study suggests that gravin is an important component of cardiac contraction regulation via increasing myofilament sensitivity to calcium. Further elucidation of the mechanism can provide insights to role of gravin if any in the pathophysiology of impaired contractility.
... 44 7 However, they rapidly undergo adaptation and stabilize at a lower PO than non-phosphorylated channels. 64 Remarkably, three expected functional outcomes of phosphorylation have been reported for channel activity: increase, 52,54 decrease, 65,66 and no effect. 67 Many of these studies have looked at the overall phosphorylation state of the channel rather than focusing on a single phosphorylation site. ...
Thesis
The Ryanodine Receptor type 2 (RyR2) the major calcium-release channel in the heart, where it is fundamental for excitation-contraction coupling, the process transducing electrical signals into mechanical contraction. The role of RyR2 dysfunction as a trigger of cardiac arrhythmia due to inherited mutations is firmly established, but the fundamental mechanisms of RyR2 regulation in normal cardiac physiology, and dysregulation in other forms of inherited and acquired heart disease remain partially understood. In this dissertation, we took advantage of three novel genetically-engineered animal models with mutations in RyR2 to better understand of the role of this ion channel in the healthy and diseased heart. First, we derived a congenic mouse line with ablation of the S2808 phosphorylation to revisit the hypothesis that this site is critical for RyR2 regulation, and sort out one of the differences between two mouse models generated by different laboratories that have fueled a long-standing controversy: the genetic background. Consistent with previous studies performed in Sv129/C57Bl6 mice, our data demonstrate that S2808A mice in the C57Bl/6 background behave like WT when subjected to acute and chronic stress. These data support the idea that S2808 phosphorylation is unlikely fundamental for RyR2 regulation during the normal adrenergic response or heart failure progression. Furthermore, they suggest that the genetic background may not be the cause for the opposing results obtained with S2808A mice from different laboratories. Second, we used a novel RyR2 knock-out rabbit model to elucidate the effects of decreased RyR2 expression on cardiac function and the possible underlying compensatory mechanisms. We show that homozygous knock-out of RyR2 is lethal, while heterozygous knock-out decreases RyR2 expression by 60% without producing an abnormal phenotype. Our data indicate that RyR2 deficiency is likely compensated by upregulation of channel function by decreasing the phosphorylation of S2031. Hence, RyR2 function is likely backed by a protein reserve, and channel deficiency is readily compensated. Nonetheless, during acute adrenergic stimulation the contraction velocity and calcium release are slower in cardiomyocytes from mutant animals, suggesting that a 40% RyR2 level may be insufficient to maintain calcium release flux during acute stress. These results give additional significance to phosphorylation of S2031, a site often ignored in the regulatory scheme of RyR2. Third, we performed a multi-level characterization of the novel mutation P1124L, identified in a patient with hypertrophic cardiomyopathy. Since this is one of a handful of RyR2 mutations associated with structural remodeling of the heart, its study may uncover novel pathogenic mechanisms of RyR2 dysregulation. We show that P1124L induces conformational changes in the SPRY2 domain of RyR2, affecting the sensitivity of the channel to cytosolic and luminal calcium. In a mouse model, P1124L produces cardiac hypertrophy and increases the susceptibility to arrhythmia. While we have yet to fully elucidate the underlying pathogenic mechanisms, these studies suggest that specific RyR2 mutations may cause cardiac hypertrophy while at the same inducing arrhythmias that are typical of previously-described mutations. These three stories share a common aim: to obtain a better understanding of RyR2 regulation in cardiac pathophysiology. From the regulatory role of phosphorylation to the clinical relevance of mutations in the RYR2 gene and the underlying pathogenic mechanisms, each model addressed in this dissertation provides novel insights into the role of RyR2 as an essential player in excitation-contraction coupling, and the possible culprit of severe cardiac disease.
... PP2A is the major serine/threonine phosphatase in eukaryotic cells and highly abundant in the heart [74]. It is involved in the dephosphorylation of several cardiac myocyte PKA substrate proteins that participate in excitation-contraction coupling, such as the L-type Ca 2+ channel [75,76], cMyBP-C [77], cTnI [78], most likely the RyR2 [25,[79][80][81] and to a lesser extent PLN [82]. Due to its substrate spectrum, PP2A activity has a decisive influence on cardiac myocyte Ca 2+ -cycling and cardiac contractility. ...
Article
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Pathologies, such as cancer, inflammatory and cardiac diseases are commonly associated with long-term increased production and release of reactive oxygen species referred to as oxidative stress. Thereby, protein oxidation conveys protein dysfunction and contributes to disease progression. Importantly, trials to scavenge oxidants by systemic antioxidant therapy failed. This observation supports the notion that oxidants are indispensable physiological signaling molecules that induce oxidative post-translational modifications in target proteins. In cardiac myocytes, the main driver of cardiac contractility is the activation of the β-adrenoceptor-signaling cascade leading to increased cellular cAMP production and activation of its main effector, the cAMP-dependent protein kinase (PKA). PKA-mediated phosphorylation of substrate proteins that are involved in excitation-contraction coupling are responsible for the observed positive inotropic and lusitropic effects. PKA-actions are counteracted by cellular protein phosphatases (PP) that dephosphorylate substrate proteins and thus allow the termination of PKA-signaling. Both, kinase and phosphatase are redox-sensitive and susceptible to oxidation on critical cysteine residues. Thereby, oxidation of the regulatory PKA and PP subunits is considered to regulate subcellular kinase and phosphatase localization, while intradisulfide formation of the catalytic subunits negatively impacts on catalytic activity with direct consequences on substrate (de)phosphorylation and cardiac contractile function. This review article attempts to incorporate the current perception of the functionally relevant regulation of cardiac contractility by classical cAMP-dependent signaling with the contribution of oxidant modification.
... A nucleotide degradation and decreased muscle ATP and ADP content have been reported. The ATP is necessary to drive the Na + /K + pump which maintains ionic gradients across the sarcolemma; resequester the Ca ++ into the cisternae; and power contraction [144][145][146]. The production of ATP can be the result of anaerobic respiration, which breaks glucose down into ATP and lactic acid, or aerobic respiration when ATP, carbon dioxide, and water are formed. ...
... [9][10][11][12] Additionally, in vitro experiments examining single channel RyR2 activity have shown that dephosphorylation of RyR2 by phosphatases might enhance channel activity, suggesting that inhibitory phosphorylation sites may decrease channel opening countering the effects of S2808 and S2814 phosphorylation. 13 Downloaded from http://ahajournals.org by on September 21, 2020 S2808 and S2814 hyperphosphorylation are seen in cAF but not pAF, 14 and it is unclear whether these events are truly driving AF or are the consequence of the rapid atrial rate and the related remodeling during AF. Furthermore, these post-translational modifications cannot explain the increase in RyR2 activity seen in pAF, as enhanced RyR2 activity has been shown in bilayer studies from human pAF samples with unchanged S2808 and S2814 phosphorylation. ...
Article
Background: Enhanced diastolic calcium (Ca ²⁺ ) release via ryanodine receptor type-2 (RyR2) has been implicated in atrial fibrillation (AF) promotion. Diastolic sarcoplasmic reticulum (SR) Ca ²⁺ leak is caused by increased RyR2 phosphorylation by protein kinase A (PKA) or Ca ²⁺ /calmodulin-dependent kinase-II (CaMKII) phosphorylation, or less dephosphorylation by protein phosphatases. However, considerable controversy remains regarding the molecular mechanisms underlying altered RyR2 function in AF. We thus sought to determine the role of 'striated muscle preferentially expressed protein kinase' (SPEG), a novel regulator of RyR2 phosphorylation, in AF pathogenesis. Methods: Western blotting was performed with right atrial biopsies from paroxysmal (p)AF patients. SPEG atrial knock-out (aKO) mice were generated using adeno-associated virus 9 (AAV9). In mice, AF inducibility was determined using intracardiac programmed electrical stimulation (PES), and diastolic Ca ²⁺ leak in atrial cardiomyocytes was assessed using confocal Ca ²⁺ imaging. Phospho-proteomics studies and western blotting were used to measure RyR2 phosphorylation. In order to test the effects of RyR2-S2367 phosphorylation, knock-in mice with an inactivated S2367 phosphorylation site (S2367A) and a constitutively activated S2367 residue (S2367D) were generated using CRISPR-Cas9. Results: Western blotting revealed decreased SPEG protein levels in atrial biopsies from pAF patients in comparison to patients in sinus rhythm. SPEG aKO mice exhibited increased susceptibility to pacing-induced AF by PES and enhanced Ca ²⁺ spark frequency in atrial cardiomyocytes with Ca ²⁺ imaging, establishing a causal role for decreased SPEG in AF pathogenesis. Phospho-proteomics in hearts from SPEG cardiomyocyte knock-out mice identified RyR2-S2367 as a novel kinase substrate of SPEG. Additionally, western blotting demonstrated that RyR2-S2367 phosphorylation was also decreased in pAF patients. RyR2-S2367A mice exhibited an increased susceptibility to pacing-induced AF as well as aberrant atrial SR Ca ²⁺ leak. In contrast, RyR2-S2367D mice were resistant to pacing-induced AF. Conclusions: Unlike other kinases (PKA, CaMKII) that increase RyR2 activity, SPEG phosphorylation reduces RyR2-mediated SR Ca ²⁺ -release. Reduced SPEG levels and RyR2-S2367 phosphorylation typified patients with pAF. Studies in S2367 knock-in mouse models showed a causal relationship between reduced S2367 phosphorylation and AF susceptibility. Thus, modulating SPEG activity and phosphorylation levels of the novel S2367 site on RyR2 may represent a novel target for AF treatment.
... Similarly, Sonnleitner et al. 33 showed using a lipid bilayer system that PP1 treated RyR1 (isolated from rabbit skeletal muscles) has decreased single-channel activity. However, in contrast to this understanding, Terentyev et al. 34 reported that PP1 actually increased diastolic RyR2 activity using myocytes permeabilized with saponin and a lipid bilayer system. Finally, Carter et al. 35 reported the paradoxical finding that both PKA phosphorylation and PP1 dephosphorylation of RyR2 increase its activity in a lipid bilayer, suggesting a non-linear relationship between phosphorylation state and activity. ...
Article
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Altered Ca(2+)-handling in atrial fibrillation (AF) has been associated with dysregulated protein phosphatase 1 (PP1) and subcellular heterogeneities in protein phosphorylation but the underlying mechanisms remain unclear. This is due to a lack of investigation into the local, rather than global, regulation of PP1 on different subcellular targets such as ryanodine receptor type-2 (RyR2), especially in AF.Methods and ResultsWe tested the hypothesis that impaired local regulation of PP1 causes RyR2 hyperphosphorylation thereby promoting AF susceptibility. To specifically disrupt PP1's local regulation of RyR2, we used the spinophilin knockout (Sp-/-) mice (Mus musculus) since PP1 is targeted to RyR2 via spinophilin. Without spinophilin, the interaction between PP1 and RyR2 was reduced by 64% while RyR2 phosphorylation was increased by 43% at Serine (S)2814 but unchanged at S2808. Lipid bilayer experiments revealed that single RyR2 channels isolated from Sp-/- hearts had an increased open probability. Likewise, Ca(2+) spark frequency normalized to sarcoplasmic reticulum Ca(2+) content was also enhanced in Sp-/- atrial myocytes but normalized by Ca(2+)/calmodulin-dependent kinase II (CaMKII) inhibitors KN-93 and AIP, and by genetic inhibition of RyR2 S2814 phosphorylation. Finally, Sp-/- mice exhibited increased atrial ectopy and susceptibility to pacing-induced AF, both of which were also prevented by the RyR2 S2814A mutation. PP1 regulates RyR2 locally by counteracting CaMKII phosphorylation of RyR2. Decreased local PP1 regulation of RyR2 contributes to RyR2 hyperactivity and promotes AF susceptibility. This represents a novel mechanism for subcellular modulation of calcium channels, and may represent a potential drug target of AF.
... Several recent studies of SR Ca 2+ release in cardiomyocytes and RyR2 gating in lipid bilayers suggest that the effect of PKA-mediated RyR2 phosphorylation on RyR2 function is more complex than previously assumed. It has been shown that RyR2 is significantly phosphorylated at Ser-2808/9 under "basal" condition (in the absence of adrenergic stimulation or PKA activation) [13,20,22,23] and reduction in RyR2 phosphorylation increases SR Ca 2+ release, causing SR Ca 2+ depletion [24]. Therefore, it seems that RyR2 dephosphorylation, rather than phosphorylation, increases the channel activity. ...
Article
Functional impact of cardiac ryanodine receptor (type 2 RyR or RyR2) phosphorylation by protein kinase A (PKA) remains highly controversial. In this study, we characterized a functional link between PKA-mediated RyR2 phosphorylation level and sarcoplasmic reticulum (SR) Ca2 + release and leak in permeabilized rabbit ventricular myocytes. Changes in cytosolic [Ca2 +] and intra-SR [Ca2 +]SR were measured with Fluo-4 and Fluo-5N, respectively. Changes in RyR2 phosphorylation at two PKA sites, serine-2031 and -2809, were measured with phospho-specific antibodies. cAMP (10 μM) increased Ca2 + spark frequency approximately two-fold. This effect was associated with an increase in SR Ca2 + load from 0.84 to 1.24 mM. PKA inhibitory peptide (PKI; 10 μM) abolished the cAMP-dependent increase of SR Ca2 + load and spark frequency. When SERCA was completely blocked by thapsigargin, cAMP did not affect RyR2-mediated Ca2 + leak. The lack of a cAMP effect on RyR2 function can be explained by almost maximal phosphorylation of RyR2 at serine-2809 after sarcolemma permeabilization. This high RyR2 phosphorylation level is likely the consequence of a balance shift between protein kinase and phosphatase activity after permeabilization. When RyR2 phosphorylation at serine-2809 was reduced to its “basal” level (i.e. RyR2 phosphorylation level in intact myocytes) using kinase inhibitor staurosporine, SR Ca2 + leak was significantly reduced. Surprisingly, further dephosphorylation of RyR2 with protein phosphatase 1 (PP1) markedly increased SR Ca2 + leak. At the same time, phosphorylation of RyR2 at serine 2031 did not significantly change under identical experimental conditions. These results suggest that RyR2 phosphorylation by PKA has a complex effect on SR Ca2 + leak in ventricular myocytes. At an intermediate level of RyR2 phosphorylation SR Ca2 + leak is minimal. However, complete dephosphorylation and maximal phosphorylation of RyR2 increases SR Ca2 + leak.
... Increased PP1 in CHF is in accordance with a previous study of this model of heart failure, as well as a dog model of coronary microembolization-induced heart failure, and has even been shown in myocardial tissue from patients with end-stage heart failure [42,43]. Interestingly, Terentyev and coworkers found PP1 to increase the open probability of RyR and increasing Ca 2+ spark frequency [44]. This was attributed to dephosphorylation of RyR. ...
Article
Abnormal cellular Ca2+ handling contributes to both contractile dysfunction and arrhythmias in heart failure. Reduced Ca2+ transient amplitude due to decreased sarcoplasmic reticulum Ca2+ content is a common finding in heart failure models. However, heart failure models also show increased propensity for diastolic Ca2+ release events which occur when sarcoplasmic reticulum Ca2+ content exceeds a certain threshold level. Such Ca2+ release events can initiate arrhythmias. In this study we aimed to investigate if both of these aspects of altered Ca2+ homeostasis could be found in left ventricular cardiomyocytes from rats with different states of cardiac function six weeks after myocardial infarction when compared to sham-operated controls. Video edge-detection, whole-cell Ca2+ imaging and confocal line-scan imaging were used to investigate cardiomyocyte contractile properties, Ca2+ transients and Ca2+ waves. In baseline conditions, i.e. without beta-adrenoceptor stimulation, cardiomyocytes from rats with large myocardial infarction, but without heart failure, did not differ from sham-operated animals in any of these aspects of cellular function. However, when exposed to beta-adrenoceptor stimulation, cardiomyocytes from both non-failing and failing rat hearts showed decreased sarcoplasmic reticulum Ca2+ content, decreased Ca2+ transient amplitude, and increased frequency of Ca2+ waves. These results are in line with a decreased threshold for diastolic Ca2+ release established by other studies. In the present study, factors that might contribute to a lower threshold for diastolic Ca2+ release were increased THR286 phosphorylation of Ca2+/calmodulin-dependent protein kinase II and increased protein phosphatase 1 abundance. In conclusion, this study demonstrates both decreased sarcoplasmic reticulum Ca2+ content and increased propensity for diastolic Ca2+ release events in ventricular cardiomyocytes from rats with heart failure after myocardial infarction, and that these phenomena are also found in rats with large myocardial infarctions without heart failure development. Importantly, beta-adrenoceptor stimulation is necessary to reveal these perturbations in Ca2+ handling after a myocardial infarction.
... Isoflurane is also known to inhibit L-type Ca 2+ channel and to decrease action potential duration, thus decreasing SR Ca 2+ release. 23,45,46 One cannot rule out that the modification of Ca 2+ transient seen after the administration of isoflurane might be partly due to a reduced Ca 2+ -induced Ca 2+ release. ...
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Background: The mitochondrial permeability transition pore (PTP) has been established as an important mediator of ischemia-reperfusion-induced cell death. The matrix protein cyclophilin D (CypD) is the best known regulator of PTP opening. Therefore, the authors hypothesized that isoflurane, by inhibiting the respiratory chain complex I, another regulator of PTP, might reinforce the myocardial protection afforded by CypD inhibition. Methods: Adult mouse or isolated cardiomyocytes from wild-type or CypD knockout (CypD-KO) mice were subjected to ischemia or hypoxia followed by reperfusion or reoxygenation. Infarct size was assessed in vivo. Mitochondrial membrane potential and PTP opening were assessed using tetramethylrhodamine methyl ester perchlorate and calcein-cobalt fluorescence, respectively. Fluo-4 AM and rhod-2 AM staining allowed the measurement, by confocal microscopy, of Ca transient and Ca transfer from sarcoplasmic reticulum (SR) to mitochondria after caffeine stimulation. Results: Both inhibition of CypD and isoflurane significantly reduced infarct size (-50 and -37%, respectively) and delayed PTP opening (+63% each). Their combination had no additive effect (n = 6/group). CypD-KO mice displayed endogenous protection against ischemia-reperfusion. Isoflurane depolarized the mitochondrial membrane (-28%, n = 5), decreased oxidative phosphorylation (-59%, n = 5), and blunted the caffeine-induced Ca transfer from SR to mitochondria (-22%, n = 7) in the cardiomyocytes of wild-type mice. Importantly, this transfer was spontaneously decreased in the cardiomyocytes of CypD-KO mice (-25%, n = 4 to 5). Conclusions: The results suggest that the partial inhibitory effect of isoflurane on respiratory complex I is insufficient to afford a synergy to CypD-induced protection. Isoflurane attenuates the Ca transfer from SR to mitochondria, which is also the prominent role of CypD, and finally prevents PTP opening.
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Protein phosphorylation is a major regulatory mechanism of signal transduction cascades in eukaryotic cells, catalysed by kinases and reversed by protein phosphatases (PPs). Sequencing of entire genomes has revealed that ~3% of all eukaryotic genes encode kinases or PPs. Surprisingly, there appear to be 2-5 times fewer PPs than kinases. Over the past two decades it has become apparent that the diversity of Ser/Thr-specific PPs (STPP) was achieved not only by the evolution of new catalytic subunits, but also by the ability of a single catalytic subunit to interact with multiple interacting proteins. PP1, a STPP, is involved in the control of important cellular mechanisms. Several isoforms of PP1 are known in mammals: PP1α, PP1β and PP1γ. The various isoforms are highly similar, except for the N- and C-termini. The current view is that since PPs possess exquisite specificities in vivo, the key control mechanism must reside in the nature of the PP1 Interacting Protein (PIP) to which they bind. An increasing number of PIPs have been identified that are responsible for regulating the catalytic activity of PPs. Indeed, the diversity of such PIPs explains the need for relatively few catalytic subunit types, and makes them attractive targets for pharmacological intervention. This review will summarize the PIPs identified using the Yeast Two Hybrid methodology and alternative techniques, for instance bioinformatic and proteomic approaches. Further, it compiles 129 PP1-PIP relevant physiological interactions that are well documented in the literature. Finally, the use of PIPs as therapeutic targets will be addressed.
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Ryanodine receptor 2 (RyR2) is an ion channel in the heart responsible for releasing into the cytosol most of the Ca²⁺ required for contraction. Proper regulation of RyR2 is critical, as highlighted by the association between channel dysfunction and cardiac arrhythmia. Lower RyR2 expression is also observed in some forms of heart disease; however, there is limited information on the impact of this change on excitation-contraction (e-c) coupling, Ca²⁺-dependent arrhythmias, and cardiac performance. We used a constitutive knock-out of RyR2 in rabbits (RyR2-KO) to assess the extent to which a stable decrease in RyR2 expression modulates Ca²⁺ handling in the heart. We found that homozygous knock-out of RyR2 in rabbits is embryonic lethal. Remarkably, heterozygotes (KO+/−) show ~50% loss of RyR2 protein without developing an overt phenotype at the intact animal and whole heart levels. Instead, we found that KO+/− myocytes show (1) remodeling of RyR2 clusters, favoring smaller groups in which channels are more densely arranged; (2) lower Ca²⁺ spark frequency and amplitude; (3) slower rate of Ca²⁺ release and mild but significant desynchronization of the Ca²⁺ transient; and (4) a significant decrease in the basal phosphorylation of S2031, likely due to increased association between RyR2 and PP2A. Our data show that RyR2 deficiency, although remarkable at the molecular and subcellular level, has only a modest impact on global Ca²⁺ release and is fully compensated at the whole-heart level. This highlights the redundancy of RyR2 protein expression and the plasticity of the e-c coupling apparatus.
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An increase in cytosolic protein phosphatases (PPs) de-phosphorylates phospholamban, decreasing the Ca(2+) uptake of the sarcoplasmic reticulum (SR). The effects of PP inhibitors on cellular Ca(2+) handling were investigated. Twitch Ca(2+) transients (CaTs) and cell shortening were measured in intact rat cardiac myocytes, and caffeine-induced Ca(2+) transients (CaffCaTs) and Ca(2+) sparks were studied in saponin-permeabilized cells. Calyculin A augmented isoproterenol-induced increases in CaTs and cell shortening without altering the diastolic [Ca(2+)](i) and twitch [Ca(2+)](i) decay. The protein kinase A catalytic subunit (PKA(cat)) increased the peak of CaffCaTs between 5 and 50 U/ml, and the addition of inhibitor-1 (I-1) augmented the increase. PKA(cat) increased Ca(2+) spark frequency and the addition of I-1 increased it further. PKA(cat) at 50 U/ml amplified the peak and prolonged the duration of Ca(2+) sparks, whereas the addition of I-1 did not alter them. An abrupt inhibition of SR Ca(2+) uptake following exposure to PKA(cat) caused a gradual decrease in Ca(2+) spark frequency, but the addition of I-1 did not accelerate the decline of Ca(2+) spark frequency or CaffCaTs. Inhibition of PPs augmented the inotropic effect of isoproterenol. Specific inhibition of PP1 could stimulate the Ca(2+) uptake of the SR with less significant effects on the Ca(2+) release.
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Phosphorylation of the cardiac ryanodine receptor (RyR2) is a key mechanism regulating sarcoplasmic reticulum (SR) Ca2+ release. Differences in opinion have arisen over the importance assigned to specific phosphorylation sites on RyR2, over the kinase (s) suggested to directly phosphorylate RyR2 and surrounding the possibility that altered phosphorylation of RyR2 is associated with contractile dysfunction observed in heart failure. Ca2+/calmodulin dependent protein kinase II (CaMKII) can phosphorylate RyR2 and modulate its activity. This phosphorylation positively modulates cardiac inotropic function but in extreme situations such as heart failure, elevated CaMKII activity can adversely increase Ca2+ release from the SR and lead to arrhythmogenesis. Although other kinases can phosphorylate RyR2, most notably cAMP-dependent protein kinase (PKA), evidence for a key role of CaMKII in mediating RyR2-dependent Ca2+ release is emerging. Future challenges include (i) fully identifying mechanisms of CaMKII interaction with the RyR2 complex and (ii) given the ubiquitous expression of CaMKII, developing selective strategies to modulate RyR2-targeted CaMKII activity and allow improved understanding of its role in normal and diseased heart.
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As the most prototypical G protein-coupled receptor, beta-adrenergic receptor (betaAR) regulates the pace and strength of heart beating by enhancing and synchronizing L-type channel (LCC) Ca(2+) influx, which in turn elicits greater sarcoplasmic reticulum (SR) Ca(2+) release flux via ryanodine receptors (RyRs). However, whether and how betaAR-protein kinase A (PKA) signaling directly modulates RyR function remains elusive and highly controversial. By using unique single-channel Ca(2+) imaging technology, we measured the response of a single RyR Ca(2+) release unit, in the form of a Ca(2+) spark, to its native trigger, the Ca(2+) sparklet from a single LCC. We found that acute application of the selective betaAR agonist isoproterenol (1 microM, < or = 20 min) increased triggered spark amplitude in an LCC unitary current-independent manner. The increased ratio of Ca(2+) release flux underlying a Ca(2+) spark to SR Ca(2+) content indicated that betaAR stimulation helps to recruit additional RyRs in synchrony. Quantification of sparklet-spark kinetics showed that betaAR stimulation synchronized the stochastic latency and increased the fidelity (i.e., chance of hit) of LCC-RyR intermolecular signaling. The RyR modulation was independent of the increased SR Ca(2+) content. The PKA antagonists Rp-8-CPT-cAMP (100 microM) and H89 (10 microM) both eliminated these effects, indicating that betaAR acutely modulates RyR activation via the PKA pathway. These results demonstrate unequivocally that RyR activation by a single LCC is accelerated and synchronized during betaAR stimulation. This molecular mechanism of sympathetic regulation will permit more fundamental studies of altered betaAR effects in cardiovascular diseases.
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Diabetes mellitus is a major risk factor for cardiovascular complications. Intracellular Ca(2+) release plays an important role in the regulation of muscle contraction. Sarcoplasmic reticulum Ca(2+) release is controlled by dedicated molecular machinery, composed of a complex of cardiac ryanodine receptors (RyR2s). Acquired and genetic defects in this complex result in a spectrum of abnormal Ca(2+) release phenotypes in heart. Cardiovascular dysfunction is a leading cause for mortality of diabetic individuals due, in part, to a specific cardiomyopathy, and to altered vascular reactivity. Cardiovascular complications result from multiple parameters, including glucotoxicity, lipotoxicity, fibrosis, and mitochondrial uncoupling. In diabetic subjects, oxidative stress arises from an imbalance between production of reactive oxygen and nitrogen species and capability of the system to readily detoxify reactive intermediates. To date, the etiology underlying diabetes-induced reductions in myocyte and cardiac contractility remains incompletely understood. However, numerous studies, including work from our laboratory, suggest that these defects stem in part from perturbation in intracellular Ca(2+) cycling. Since the RyR2s are one of the well-characterized redox-sensitive ion channels in heart, this article summarizes recent findings on redox regulation of cardiac Ca(2+) transport systems and discusses contributions of redox regulation to pathological cardiac function in diabetes.
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The ryanodine receptor (RyR) is an intracellular calcium release channel located on the sarco(endo)plasmic reticulum of muscle and non-muscle cells. The functional channel is composed of four identical subunits of approximately 560 kDa, which combine to form a high-conductance cation-permeable protein pore. There are three mammalian RyR isoforms that have a wide tissue expression. Their highest levels are in striated muscles where they mediate the release of stored Ca2+ leading to a rise in intracellular Ca2+ concentration and muscle contraction. Channel activity is regulated by Ca2+, Mg2+, ATP and post-translational modifications, i.e. oxidation/reduction and phosphorylation. In addition, the RyR is regulated by intramolecular protein–protein interactions, as well as by interacting with numerous accessory proteins including the dihydropyridine receptor (DHPR), FK506-binding protein (FKBP), calmodulin (CaM), sorcin and calsequestrin (CSQ). Inherited or acquired defective channel regulation results in abnormal Ca2+ handling and leads to neuromuscular disorders and arrhythmogenic cardiac disease.
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Cardiac arrhythmias can follow disruption of the normal cellular electrophysiological processes underlying excitable activity and their tissue propagation as coherent wavefronts from the primary sinoatrial node pacemaker, through the atria, conducting structures and ventricular myocardium. These physiological events are driven by interacting, voltage-dependent, processes of activation, inactivation, and recovery in the ion channels present in cardiomyocyte membranes. Generation and conduction of these events are further modulated by intracellular Ca²⁺ homeostasis, and metabolic and structural change. This review describes experimental studies on murine models for known clinical arrhythmic conditions in which these mechanisms were modified by genetic, physiological, or pharmacological manipulation. These exemplars yielded molecular, physiological, and structural phenotypes often directly translatable to their corresponding clinical conditions, which could be investigated at the molecular, cellular, tissue, organ, and whole animal levels. Arrhythmogenesis could be explored during normal pacing activity, regular stimulation, following imposed extra-stimuli, or during progressively incremented steady pacing frequencies. Arrhythmic substrate was identified with temporal and spatial functional heterogeneities predisposing to reentrant excitation phenomena. These could arise from abnormalities in cardiac pacing function, tissue electrical connectivity, and cellular excitation and recovery. Triggering events during or following recovery from action potential excitation could thereby lead to sustained arrhythmia. These surface membrane processes were modified by alterations in cellular Ca²⁺ homeostasis and energetics, as well as cellular and tissue structural change. Study of murine systems thus offers major insights into both our understanding of normal cardiac activity and its propagation, and their relationship to mechanisms generating clinical arrhythmias.
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Pathological stress including myocardial infarction and hypertension causes a negative effect on calcium regulation and homeostasis. Nevertheless, few studies reveal that Ca(2+) regulatory genes are related to pathological status in cardiomyocytes under early hypoxia. To determine the alteration of Ca(2+)-related gene in hypoxic myocytes, primary neonatal rat ventricular cardiomyocytes (NRVCMs) was isolated. Survival of hypoxic NRVCMs was significantly decreased in 6 h. We confirmed an increase of reactive oxygen species (ROS) generation and Ca(2+) overload in hypoxic NRVCMs by using 2',7'-dichlorodihydro-fluorescein diacetate (H2DCFDA) and FACS analysis. Furthermore, survival/apoptotic signals were also regulated in same condition. The expression profiles of more than 30,000 genes from NRVCMs that were subjected to early hypoxia revealed 630 genes that were differentially regulated. The intracellular Na(+) overload and Ca(2+) handling genes with at least two-fold changes were confirmed. The levels of Ca(2+)-handling proteins (calsequestrin, calmodulin, and calreticulin), ion channels (NCX, Na(+)-K(+)-ATPase, SERCA2a, and PLB), and stress markers (RyR2, ANP, and BNP) were significantly altered in early hypoxia. These results demonstrate that early hypoxia alters Ca(2+)-related gene expression in NRVCMs, leading to pathological status.
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Key points: Increased protein phosphatase 1 (PP-1) activity has been found in end stage human heart failure. Although PP-1 has been extensively studied, a detailed understanding of its role in the excitation-contraction coupling mechanism, in the normal and diseased heart, remains elusive. The present work investigates the functional effect of the PP-1 activity on local Ca2+ release events in ventricular cardiomyocytes, by using an activating peptide (PDP3) for the stimulation of the endogenous PP-1 protein. We report that acute de-phosphorylation may increase the sensitivity of RyR2 channels to Ca2+ in situ, and that the RyR2-serine2808 phosphorylation site may mediate such a process. Our approach unmasks the functional importance of the PP-1 in the regulation of the RyR2 activity, suggesting a potential role in the generation of a pathophysiological sarcoplasmic reticulum Ca2+ leak in diseased heart. Abstract: Changes in cardiac ryanodine receptor (RyR2) phosphorylation are thought to be important regulatory and disease related post-translational protein modifications. The extent of RyR2 phosphorylation is mainly determined by the balance of the activities of protein kinases and phosphatases, respectively. Increased protein phosphatase-1 (PP-1) activity has been observed in heart failure, but the regulatory role of this enzyme on intracellular Ca2+ handling remains poorly understood. To determine the physiological and pathophysiological significance of increased PP-1 activity, we investigated how the PP-1 catalytic subunit (PP-1c) alters Ca2+ sparks in permeabilized cardiomyocytes and we also applied a PP-1-disrupting peptide (PDP3) to specifically activate endogenous PP-1, including the one anchored on the RyR2 macromolecular complex. We compared wild-type (WT) and transgenic mice in which the usually highly phosphorylated site RyR2-S2808 has been ablated to investigate its involvement in RyR2 modulation (S2808A+/+ ). In WT myocytes, PP-1 increased Ca2+ spark frequency (CaSpF) by 2-fold, followed by depletion of the sarcoplasmic reticulum (SR) Ca2+ store. Similarly, PDP3 transiently increased spark frequency and decreased SR Ca2+ load. RyR2 Ca2+ sensitivity, which was assessed by Ca2+ spark recovery analysis, was increased in the presence of PDP3 when compared with a negative control peptide. S2808A+/+ cardiomyocytes did not respond to both PP-1c and PDP3 treatment. Our results suggest an increased Ca2+ sensitivity of the RyR2 upon de-phosphorylation by PP-1. Furthermore, we confirmed the S2808 site as target for PP-1 and as a potential link between the RyR2s modulation and the cellular response. This article is protected by copyright. All rights reserved.
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• We carried out confocal Ca2+ imaging in myocytes permeabilized with saponin in ‘internal’ solutions containing: MgATP, EGTA and fluo-3 potassium salt. • Permeabilized myocytes exhibited spontaneous Ca2+ sparks and waves similar to those observed in intact myocytes loaded with fluo-3 AM. • In the presence of ‘low’[EGTA] (0·05 mm), Ca2+ waves arose regularly, even at relatively low [Ca2+] (50–100 nm, free). Increasing [EGTA] resulted in decreased frequency and propagation velocity of Ca2+ waves. Propagating waves were completely abolished at [EGTA] > 0·3 mm. • The frequency of sparks increased as a function of [Ca2+] (50–400 nmrange) with no sign of a high affinity Ca2+-dependent inactivation process. • The rate of occurrence of Ca2+ sparks was increased by calmodulin and cyclic adenosine diphosphate-ribose (cADPR).
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Ryanodine receptors have recently been shown to be the Ca2+ release channels of sarcoplasmic reticulum in both cardiac muscle and skeletal muscle. Several regulatory sites are postulated to exist on these receptors, but to date, none have been definitively identified. In the work described here, we localize one of these sites by showing that the cardiac isoform of the ryanodine receptor is a preferred substrate for multifunctional Ca2+/calmodulin-dependent protein kinase (CaM kinase). Phosphorylation by CaM kinase occurs at a single site encompassing serine 2809. Antibodies generated to this site react only with the cardiac isoform of the ryanodine receptor, and immunoprecipitate only cardiac [3H]ryanodine-binding sites. When cardiac junctional sarcoplasmic reticulum vesicles or partially purified ryanodine receptors are fused with planar bilayers, phosphorylation at this site activates the Ca2+ channel. In tissues expressing the cardiac isoform of the ryanodine receptor, such as heart and brain, phosphorylation of the Ca2+ release channel by CaM kinase may provide a unique mechanism for regulating intracellular Ca2+ release.
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Isoprenaline stimulation of perfused rabbit hearts was associated with simultaneous phosphorylation of proteins in the myofilaments and phospholamban in the sarcoplasmic reticulum (SR). Hearts were perfused with Krebs-Henseleit buffer containing [32P]Pi, freeze-clamped in a control condition or at the peak of the inotropic response to isoprenaline, and myofibrils and SR were prepared from the same hearts. Stimulation of 32P incorporation in troponin I (TnI) and C-protein by isoprenaline was associated with a decrease in Ca2+-sensitivity of the myofibrillar Mg2+-dependent ATPase activity. Stimulation of 32P incorporation in SR by isoprenaline was associated with an increase in the initial rates of oxalate-facilitated Ca2+ transport, assayed with SR vesicles in either microsomal fractions or homogenates from the perfused hearts. These findings provide evidence that phosphorylation of TnI, C-protein and phospholamban in the intact cell is associated with functional alterations of the myofibrils and SR which may be responsible in part for the effects of catecholamines on the mammalian myocardium.
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The voltage dependence of the intracellular Ca2+ transients was measured in single rat ventricular myocytes with the fluorescent Ca2+ indicator dye fura-2. The whole-cell voltage clamp technique was used to measure the membrane current, and 0.9 mM fura-2 was loaded into the cell by including it in the dialyzing solution of the patch electrode. A mechanical light chopper operating at 1200 Hz was used to obtain simultaneous measurements of the intracellular Ca2+ activity with fluorescence excitation on either side of the isosbestic point (330 nm and 410 nm). The symmetry of the two optical Ca2+ signals was used as a criterion to guard against artifacts resulting, for instance, from motion. The voltage dependence of peak Ca2+ current and the Ca2+ transient measured 25 ms after depolarizing clamps from a holding potential of -40 mV were bell-shaped and virtually identical. The Ca2+ entry estimated from the integral of the Ca2+ current (0 mV, 25 ms) corresponds to a 5-10 microM increase in the total intracellular Ca2+ concentration, whereas the optical signal indicated a 100 microM increase in total intracellular Ca2+. Repolarization of clamp pulses from highly positive potentials were accompanied by a second Ca2+ transient, the magnitude of which, when summed with that measured during depolarization, was nearly constant. Ryanodine (10 microM) had little or no effect on the peak Ca2+ current but reduced the magnitude of the early Ca2+ transients by 70-90%. Epinephrine (1 microM) increased the Ca2+ current and the Ca2+ transients, accelerated the rate of decline of the Ca2+ transients at potentials between -30 and +70 mV, and reduced the intracellular [Ca2+] below baseline at potentials positive to +80 or negative to -40 mV, where clamp pulses did not elicit any Ca2+ release. Elevation of intracellular cAMP mimicked the relaxant effect of epinephrine at depolarizing potentials, whereas elevation of extracellular [Ca2+] did not. These results suggest that most of the activator Ca2+ in rat ventricular cells is released from the sarcoplasmic reticulum as a graded response to sarcolemmal Ca2+ influx. Consistent with a graded Ca2+-induced Ca2+ release we find that epinephrine increases the internal Ca2+ release by increasing the Ca2+ current. Epinephrine may also increase the Ca2+ content of the sarcoplasmic reticulum that may, in turn, increase the Ca2+-induced Ca2+ release. The relaxant effect of epinephrine appears to be caused by enhanced rate of Ca2+ resequestration and is mediated by adenylate cyclase system.
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Channel adaptation is a fundamental feature of sarcoplasmic reticulum calcium release channels (called ryanodine receptors, RyRs). It permits successive increases in the intracellular concentration of calcium (Ca2+) to repeatedly but transiently activate channels. Adaptation of RyRs in the absence of magnesium (Mg2+) and adenosine triphosphate is an extremely slow process (taking seconds). Photorelease of Ca2+ from nitrophenyl-EGTA, a photolabile Ca2+ chelator, demonstrated that RyR adaptation is rapid (milliseconds) in canine heart muscle when physiological Mg2+ concentrations are present. Phosphorylation of the RyR by protein kinase A increased the responsiveness of the channel to Ca2+ and accelerated the kinetics of adaptation. These properties of the RyR from heart may also be relevant to other cells in which multiple agonist-dependent triggering events regulate cellular functions.
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The cardiac calcium release channel (CRC) of sarcoplasmic reticulum vesicles was incorporated into planar lipid membranes to evaluate modulation of channel activity by phosphorylation and dephosphorylation. For this purpose a microsyringe application directly to the membrane was used to achieve sequential and multiple treatments of channels with highly purified kinases and phosphatases. Cyclic application of protein kinase A (PKA) or Ca/calmodulin-dependent protein kinase II (CalPK) and potato acid phosphatase or protein phosphatase 1 revealed a channel block by Mg (mM), that is referable to dephosphorylated states of the channel, and that the Mg block could be removed by phosphorylation of the CRC by either PKA or CalPK. By contrast, activation of endogenous CalPK (end CalPK) led to channel closure which could be reversed by dephosphorylation using potato acid phosphatase or protein phosphatase 1. Calmodulin by itself (which activates end CalPK in the presence of MgATP) blocks the channel in the dephosphorylated state, which can be overcome by treatment with CalPK but not PKA. Our findings reveal important insights regarding channel regulation of the ryanodine receptor: 1) the calcium release channel must be phosphorylated to be in the active state at conditions approximating physiological Mg concentrations (mM); and 2) there are multiple sites of phosphorylation on the calcium release channel with different functional consequences, which may be relevant to the regulation of E-C coupling. Phosphorylation of the CRC may be involved in recruitment of active channels, and/or it may be directly involved in each Ca contraction cycle of the heart. For example, Ca release may require phosphorylation of the CRC by protein kinases at sites which overcome the block by Mg. Inactivation may involve CRC block by calmodulin and/or phosphorylation by endogenous CalPK at the junctional face membrane.
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The amount of Ca2+ released from the sarcoplasmic reticulum (SR) is a principal determinant of cardiac contractility. Normally, the SR Ca2+ stores are mobilized through the mechanism of Ca2+-induced Ca2+ release (CICR). In this process, Ca2+ enters the cell through plasmalemmal voltage-dependent Ca2+ channels to activate the Ca2+ release channels in the SR membrane. Consequently, the control of Ca2+ release by cytosolic Ca2+ has traditionally been the main focus of cardiac excitation-contraction (EC) coupling research. Evidence obtained recently suggests that SR Ca release is controlled not only by cytosolic Ca2+, but also by Ca2+ in the lumen of the SR. The presence of a luminal Ca2+ sensor regulating release of SR luminal Ca2+ potentially has profound implications for our understanding of EC coupling and intracellular Ca2+ cycling. Here we review evidence, obtained using in situ and in vitro approaches, in support of such a luminal Ca2+ sensor in cardiac muscle. We also discuss the role of control of Ca2+ release channels by luminal Ca2+ in termination and stabilization of CICR, as well as in shaping the response of cardiac myocytes to various inotropic influences and diseased states such as Ca2+ overload and heart failure.
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Ryanodine receptor (RyR) phosphorylation by protein kinase A (PKA) may be important in modulating resting sarcoplasmic reticulum (SR) Ca2+ release, especially in heart failure. However, clear cellular data on PKA-dependent modulation of cardiac RyRs is limited because of difficulty in distinguishing between PKA effects on RyR, phospholamban (PLB), and Ca2+ current. To clarify this, we measured resting Ca2+ sparks in streptolysin-O permeabilized ventricular myocytes from wild-type (WT) and PLB knockout (PLB-KO) mice and transgenic mice expressing only double-mutant PLB (PLB-DM) that lacks the regulatory phosphorylation sites (S16A/T17A). In WT myocytes, cAMP dramatically increased Ca2+ spark frequency (CaSpF) by 2- and 3-fold when [Ca2+] was clamped at 50 and 10 nmol/L (and the SR Ca2+ content also rose by 40% and 50%). However, in PLB-KO and PLB-DM, neither CaSpF nor SR Ca2+ load was changed by the addition of 10 μmol/L cAMP (even with phosphatase inhibition). PKA activation also increased Ca2+ spark amplitude, duration, and width in WT, but not in PLB-KO or PLB-DM. RyR phosphorylation was confirmed by measurements of 32P incorporation on immunoprecipitated RyR. In intact resting myocytes, PKA activation increased CaSpF 2.8-fold in WT, but not in PLB-KO, confirming results in permeabilized myocytes. We conclude that the PKA-dependent increase in myocyte CaSpF and size is entirely attributable to PLB phosphorylation and consequent enhanced SR Ca2+ load. PKA does not seem to have any appreciable effect on resting RyR function in these ventricular myocytes. Moreover, the data provide compelling evidence that elevated intra-SR [Ca2+] increases RyR gating independent of cytosolic [Ca2+] (which was clamped).
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• To define the sub-cellular mechanisms of modulation of cardiac excitation-contraction (E-C) coupling by the -adrenergic pathway, we carried out confocal Ca2+ imaging in conjunction with recordings of inward Ca2+ current in fluo-3-loaded patch-clamped rat ventricular myocytes. • Isoproterenol (isoprenaline; ISO) increased the amplitude of the inward Ca2+ current and the globally measured intracellular Ca2+ transients. The gain of calcium-induced calcium release (CICR) was increased at all membrane potentials but especially at positive membrane potentials (> +30 mV). ISO dramatically broadened the bell-shaped voltage dependence of intracellular Ca2+ transients by shifting the descending portion of the curve to very high membrane potentials. • The number of local release events (solitary sparks and conglomerates of overlapping sparks) induced by depolarizing steps to +30 mV was increased significantly by ISO. This potentiation of events was due to increased trigger calcium current (ICa) as well the enhanced ability of ICa to induce release. The amplitude and duration of solitary sparks were increased in the presence of ISO. In addition, ISO dramatically increased the proportion and the size (‘mass’) of clustered events. • Exclusion of Na+ from the intra- and extracellular solutions prevented ISO from enhancing the ability of ICa to trigger sparks. • We conclude that -adrenergic stimulation enhances the gain of the CICR cascade by increasing the fidelity of dihydropyridine receptor (DHPR)-ryanodine receptor (RyR) coupling and by promoting cross-activation of RyRs in neighbouring release sites. Reverse Na+-Ca2+ exchange (NCX) appears to play a role in the -adrenergic enhancement of CICR by effectively contributing to the Ca2+ trigger.
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Excitation-Ca2+ release coupling is a pivotal event in cardiac function. During membrane depolarization, sarcolemmal Ca2+ influx through the voltage-operated L-type Ca2+ channels triggers the sarcoplasmic reticulum (SR) to release Ca2+ via the Ca2+-induced Ca2+ release (CICR) mechanism (Fabiato, 1983). The resultant cytosolic Ca2+ transient activates myofilament proteins and initiates contraction. Cardiac relaxation ensues when the elevated cytoplasmic Ca2+ is resequestered by the SR Ca2+ pump, or, to a lesser extent, extruded via the sarcolemmal Na+-Ca2+ exchanger. In order to meet the body's variable circulatory demands, cardiac excitation-contraction (EC) coupling is constantly modulated by an array of physiological mechanisms. Driven by sympathetic neurotransmitters and adrenal hormones, β-adrenergic receptor (βAR) signalling interacts with virtually all constituents of the EC coupling cascade, including the L-type Ca2+ channel current (ICa) (Tsien et al. 1986; Xiao & Lakatta, 1993), SR Ca2+ release channels/ryanodine receptors (RyRs) (Yoshida et al. 1992; Valdivia et al. 1995), phospholamban (PLB) (Lindemann et al. 1983; Kuschel et al. 1999a), and contractile myofilaments (Rapundalo et al. 1989; Kuschel et al. 1999b). Additionally, β-adrenergic stimulation may modulate other sarcolemmal ionic currents, such as K+ currents (Scamps, 1996), and electrogenic transporters, such as Na+-Ca2+ exchanger (in non-mammalian species) (Shuba et al. 1998), to alter the action potential configuration, and subsequently, the Ca2+ fluxes during a cardiac cycle.
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• The effects of tetracaine were studied on voltage-clamped rat ventricular myocytes, which exhibited Ca2+ overload as identified by supontaneous Ca2+ release from the sarcoplasmic reticulum (SR) as shown by the associated contractions. This Ca2+ release was initially abolished by tetracaine before returning at a lower frequency, but greater amplitude, than the control. On removal of tetracaine, there was a burst of supontaneous Ca2+ release activity. All these effects were dose dependent, from 25 to 200 mtetracaine. • The supontaneous Ca2+ release activated an inward Na+–Ca2+ exchange current as Ca2+ was pumped out of the cell. The integral of this current (i.e. the Ca2+ efflux) was increased in the presence of tetracaine. The calcium efflux per unit time was unaffected by tetracaine. • The SR Ca2+ content was increased by tetracaine, as shown by the integral of the caffeine-evoked Na+–Ca2+ exchange current. The increase of SR Ca2+ content was equal to the extra Ca2+ lost from the cell during the burst on removal of tetracaine, and to estimates of the extra calcium gained over the quiescent period following addition of tetracaine. • It is concluded that partial inhibition of calcium-induced calcium release increases SR Ca2+ content. In the steady state, cell Ca2+ balance is maintained as the lower frequency of supontaneous release (that activates efflux) is compensated for by their greater size.
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Canine cardiac sarcoplasmic reticulum is phosphorylated by adenosine 3,5-monophosphate (cAMP)-dependent and by calcium calmodulin-dependent protein kinases on a 27 000 proteolipid, called phospholamban. Both types of phosphorylation are associated with an increase in the initial rates of Ca2+ transport by SR vesicles which reflects an increased turnover of elementary steps of the calcium ATPase reaction sequence. The stimulatory effects of the protein kinases on the calcium pump may be reversed by an endogenous protein phosphatase, which can dephosphorylate both the CAMP-dependent and the calcium calmodulin-dependent sites on phospholamban. Thus, the calcium pump in cardiac sarcoplasmic reticulum appears to be under reversible regulation mediated by protein kinases and protein phosphatases.
Article
Canine cardiac sarcoplasmic reticulum vesicles contain intrinsic protein phosphatase activity, which can dephosphorylate phospholamban and regulate calcium transport. This phosphatase has been suggested to be a mixture of both type 1 and type 2 enzymes (E. G. Kranias and J. Di Salvo, 1986, J. Biol. Chem. 261, 10,029-10,032). In the present study the sarcoplasmic reticulum phosphatase activity was solubilized with n-octyl-beta-D-glucopyranoside and purified by sequential chromatography on DEAE-Sephacel, polylysine-agarose, heparin-agarose, and DEAE-Sephadex. A single peak of phosphatase activity was eluted from each column and it was coincident for both phospholamban and phosphorylase a, used as substrates. The partially purified phosphatase could dephosphorylate the sites on phospholamban phosphorylated by either cAMP-dependent or calcium-calmodulin-dependent protein kinase(s). Enzymatic activity was inhibited by inhibitor-2 and by okadaic acid (I50 = 10-20 nM), using either phosphorylase a or phospholamban as substrates. The sensitivity of the phosphatase to inhibitor-2 or okadaic acid was similar for the two sites on phospholamban, phosphorylated by the cAMP-dependent and the calcium-calmodulin-dependent protein kinases. Phospholamban phosphatase activity was enhanced (40%) by Mg2+ or Mn2+ (3 mM) while Ca2+ (0.1-10 microM) had no effect. These characteristics suggest that the phosphatase associated with cardiac sarcoplasmic reticulum is a type 1 enzyme, and this activity may participate in the regulation of Ca2+ transport through dephosphorylation of phospholamban in cardiac muscle.
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We studied beta-adrenergic agonist-stimulated phosphorylation of the ryanodine receptor in rat cardiac myocytes. The ryanodine receptor solubilized from myocytes and immunoprecipitated by a monoclonal antibody against canine cardiac ryanodine receptor was phosphorylated by the catalytic subunit of cAMP-dependent protein kinase (PKA). Incubation of saponin-permeabilized myocytes with [gamma-32P]ATP also induced ryanodine receptor phosphorylation, which was enhanced significantly in the presence of isoproterenol. This stimulating action of isoproterenol was suppressed by the beta-adrenergic antagonist, propranolol. On the other hand, exogenously added cAMP caused a much larger stimulation of phosphorylation of the ryanodine receptor in permeabilized myocytes. The beta-agonist-induced phosphorylation of the ryanodine receptor was also observed in intact myocytes from the newborn rat heart. These results suggest that the ryanodine receptor is phosphorylated by PKA during beta-adrenergic stimulation of cardiac myocytes.
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The protein phosphatases which dephosphorylate native, sarcoplasmic reticulum (SR)-associated phospholamban were studied in cardiac muscle extracts and in a Triton fraction prepared by detergent extraction of myofibrils, the latter fraction containing 70–80% of the SR-associated proteins present in the tissue. At physiological concentrations of free Mg²⁺ (1 mM), protein phosphatase 1 (PP1) accounted for approximately 70% of the total phospholamban phosphatase activity in these fractions towards either Ser-16 (the residue labelled by cAMP-dependent protein kinase, PK-A) or Thr-17 (the residue phosphorylated by an SR-associated Ca²⁺/calmodulindependent protein kinase). Protein phosphatase 2A (PP2A) and protein phosphatase 2C (PP2C) accounted for the remainder of the activity.
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A system is described that can simultaneously record cytosolic Ca2+ concentration ([Ca2+]i), cell length, and either membrane potential or current in single cardiac myocytes loaded with the fluorescent Ca2+ indicator indo-1. Fluorescence is excited by epi-illumination with 3.8-microsecond flashes of 350 +/- 5 nm light from a xenon arc. Indo-1 fluoresence is measured simultaneously in spectral windows of 391-434 nm and 457-507 nm, and the ratio of indo-1 emission in the two bands is computed as a measure of [Ca2+]i for each flash. With cells loaded with the permeant acetoxymethyl ester of indo-1, quantitation of [Ca2+]i is not precise, owing to subcellular compartmentation of indo-1; however, the instrument would allow full quantitation if indo-1 free acid was introduced by microinjection. Simultaneously, cell length is measured on-line from the bright-field image of the cell. Because fluorescence collection is time gated during the brief flash, and red light (650-750 nm) is used for the bright-field image, cell length and [Ca2+]i measurements are obtained simultaneously without cross talk. Membrane potential or current can be recorded simultaneously with indo-1 fluorescence and cell length via standard patch-clamping techniques.
Article
Calyculin A and okadaic acid induce contraction in smooth muscle fibers. Okadaic acid is an inhibitor of phosphatase activity and the aims of this study were to determine if calyculin A also inhibits phosphatase and to screen effects of both compounds on various phosphatases. Neither compound inhibited acid or alkaline phosphatases, nor the phosphotyrosine protein phosphatase. Both compounds were potent inhibitors of the catalytic subunit of type-2A phosphatase, with IC50 values of 0.5 to 1 nM. With the catalytic subunit of protein phosphatase type-1, calyculin A was a more effective inhibitor than okadaic acid, IC50 values for calyculin A were about 2 nM and for okadaic acid between 60 and 500 nM. The endogenous phosphatase of smooth muscle myosin B was inhibited by both compounds with IC50 values of 0.3 to 0.7 nM and 15 to 70 nM, for calyculin A and okadaic acid, respectively. The partially purified catalytic subunit from myosin B had IC50 values of 0.7 and 200 nM for calyculin A and okadaic acid, respectively. The pattern of inhibition for the phosphatase in myosin B therefore is similar to that of the type-1 enzyme.
Article
Microprocessor-controlled changes of [free Ca2+] at the outer surface of the sarcoplasmic reticulum (SR) wrapped around individual myofibrils of a skinned canine cardiac Purkinje cell and aequorin bioluminescence recording were used to study the mechanism of Ca2+-induced release of Ca2+ from the SR. This Ca2+ release is triggered by a rapid increase of [free Ca2+] at the outer surface of the SR of a previously quiescent skinned cell. Ca2+-induced release of Ca2+ occurred under conditions that prevented any synthesis of ATP from ADP, was affected differentially by interventions that depressed the SR Ca2+ pump about equally, and required ionic conditions incompatible with all known Ca2+-releasing, uncoupled, partial reactions of the Ca2+ pump. Increasing the [free Ca2+]trigger up to an optimum increased the amount of Ca2+ released. A supraoptimum increase of [free Ca2+] trigger inactivated Ca2+-induced release of Ca2+, but partial inactivation was also observed at [free Ca2+] below that necessary for its activation. The amplitude of the Ca2+ release induced by a given increase of [free Ca2+] decreased when the rate of this increase was diminished. These results suggest that Ca2+-induced release of Ca2+ is through a channel across the SR membrane with time- and Ca2+-dependent activation and inactivation. The inactivating binding site would have a higher affinity for Ca2+ but a lower rate constant than the activating site. Inactivation appeared to be a first-order kinetic reaction of Ca2+ binding to a single site at the outer face of the SR with a Q10 of 1.68. The removal of inactivation was the slowest step of the cycle, responsible for a highly temperature-dependent (Q10 approximately 4.00) refractory period.
Article
The phosphorylation of canine cardiac and skeletal muscle ryanodine receptors by the catalytic subunit of cAMP-dependent protein kinase has been studied. A high-molecular-weight protein (Mr 400,000) in cardiac microsomes was phosphorylated by the catalytic subunit of cAMP-dependent protein kinase. A monoclonal antibody against the cardiac ryanodine receptor immunoprecipitated this phosphoprotein. In contrast, high-molecular-weight proteins (Mr 400,000-450,000) in canine skeletal microsomes isolated from extensor carpi radialis (fast) or superficial digitalis flexor (slow) muscle fibers were not significantly phosphorylated. In agreement with these findings, the ryanodine receptor purified from cardiac microsomes was also phosphorylated by cAMP-dependent protein kinase. Phosphorylation of the cardiac ryanodine receptor in microsomal and purified preparations occurred at the ratio of about one mol per mol of ryanodine-binding site. Upon phosphorylation of the cardiac ryanodine receptor, the levels of [3H]ryanodine binding at saturating concentrations of this ligand increased by up to 30% in the presence of Ca2+ concentrations above 1 microM in both cardiac microsomes and the purified cardiac ryanodine receptor preparation. In contrast, the Ca2+ concentration dependence of [3H]ryanodine binding did not change significantly. These results suggest that phosphorylation of the ryanodine receptor by cAMP-dependent protein kinase may be an important regulatory mechanism for the calcium release channel function in the cardiac sarcoplasmic reticulum.
Article
Insulin-induced membrane changes were investigated in K(+)-depleted rat muscle. Male Sprague-Dawley rats were placed on a K(+)-free but otherwise adequate diet for 5-8 wk; serum K+ concentration ([K+]) dropped to 1.2-3.2 mM. Omohyoid membrane potential was -81 mV in 5.5 mM [K+] (SO4(2-)). Exposure to either insulin or low (0.5 mM) [K+] singly changed potential only slightly. The combination resulted in depolarization of 90% of fibers (-43 mV) and hyperpolarization of 10% of fibers (-101 mV). Fibers from normokalemic rats did not depolarize. Tetrodotoxin (TTX) blocked depolarization, implying the presence of noninactivating TTX-sensitive Na+ channels. K+ currents were measured using the three-electrode voltage clamp; movement of other ions was prevented by ion substitution, channel blockers, and depolarization-induced channel inactivation. K+ conductance was similar in control fibers with or without insulin. In the absence of insulin, currents in K(+)-depleted fibers were offset by a large leakage current that was significantly diminished when insulin was present. The insulin-induced current decrease was observed in nitrendipine, suggesting that the apparent decreased outward current was not an inward current carried by Ca2+. Data are consistent with altered Na+ and K+ channels in K(+)-depleted muscle, i.e., insulin-related closing of K+ channels initiates depolarization, which is then sustained by opening of noninactivating Na+ channels.