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Background In experimental heart failure animal models, remodeling of skeletal and cardiac muscle ryanodine receptors (RyR), including phosphorylation, S -nitrosylation and oxidation, have been reported to contribute to pathologic Ca 2+ release, impaired muscle function and fatigue. However, it is not known whether similar remodeling of RyR1 in skeletal muscle occurs in patients with heart failure, and if this is associated with impairment of physical activity. Methods We studied 8 sedentary patients with New York Heart Association (NYHA) Class III heart failure and 7 age-matched, healthy, but sedentary controls. All heart failure patients had NYHA Class III and peak VO 2 , echocardiography and NT-proBNP data consistent with moderate to severe heart failure. The age-matched controls included were allowed hypertension but sub-clinical heart failure was to have been ruled out by normal peak VO 2 , echocardiography and NT-proBNP. Results Exercise capacity (VO 2 max) differed by almost 2-fold between heart failure patients and age-matched controls. Compared with controls, skeletal muscle RyR1 in heart failure patients was excessively phosphorylated, S -nitrosylated and oxidized. Furthermore, RyR1 from heart failure patients was depleted of its stabilizing protein FK 506–binding protein 12 (FKBP12, or calstabin1). Conclusions For the first time we show that skeletal muscle RyR1 from human heart failure is post-translationally modified, which corroborates previous data from experimental animal studies. This indicates pathologic Ca 2+ release as a potential mechanism behind skeletal muscle weakness and impaired exercise tolerance in patients with heart failure and suggests a potential target for pharmacologic intervention.

Summary Age-related loss of muscle mass and force (sarcopenia) contributes to disability and increased mortality. Ryanodine receptor 1 (RyR1) is the skeletal muscle sarcoplasmic reticulum calcium release channel required for muscle contraction. RyR1 from aged (24 months) rodents was oxidized, cysteine-nitrosylated, and depleted of the channel-stabilizing subunit calstabin1, compared to RyR1 from younger (3–6 months) adults. This RyR1 channel complex remodeling resulted in "leaky" channels with increased open probability, leading to intracellular calcium leak in skeletal muscle. Similarly, 6-month-old mice harboring leaky RyR1-S2844D mutant channels exhibited skeletal muscle defects comparable to 24-month-old wild-type mice. Treating aged mice with S107 stabilized binding of calstabin1 to RyR1, reduced intracellular calcium leak, decreased reactive oxygen species (ROS), and enhanced tetanic Ca 2+ release, muscle-specific force, and exercise capacity. Taken together, these data indicate that leaky RyR1 contributes to age-related loss of muscle function.

SummaryPhosphodiesterases (PDEs) regulate the local concentration of 3′,5′ cyclic adenosine monophosphate (cAMP) within cells. cAMP activates the cAMP-dependent protein kinase (PKA). In patients, PDE inhibitors have been linked to heart failure and cardiac arrhythmias, although the mechanisms are not understood. We show that PDE4D gene inactivation in mice results in a progressive cardiomyopathy, accelerated heart failure after myocardial infarction, and cardiac arrhythmias. The phosphodiesterase 4D3 (PDE4D3) was found in the cardiac ryanodine receptor (RyR2)/calcium-release-channel complex (required for excitation-contraction [EC] coupling in heart muscle). PDE4D3 levels in the RyR2 complex were reduced in failing human hearts, contributing to PKA-hyperphosphorylated, “leaky” RyR2 channels that promote cardiac dysfunction and arrhythmias. Cardiac arrhythmias and dysfunction associated with PDE4 inhibition or deficiency were suppressed in mice harboring RyR2 that cannot be PKA phosphorylated. These data suggest that reduced PDE4D activity causes defective RyR2-channel function associated with heart failure and arrhythmias.

ObjectivesWe hypothesized that some aspects of left ventricular assist device (LVAD) reverse remodeling could be independent of hemodynamic factors and would primarily depend upon normalization of neurohormonal milieu.BackgroundThe relative contributions of LVAD-induced hemodynamic unloading (provided to the left ventricle [LV]) and normalized neurohormonal milieu (provided to LV and right ventricle [RV]) to reverse remodeling are not understood.MethodsStructural and functional characteristics were measured from hearts of 65 medically managed transplant patients (MED), 30 patients supported with an LVAD, and 5 nonfailing donor hearts not suitable for transplantation.ResultsCompared with MED patients, diastolic pulmonary pressures trended lower (p < 0.01) and cardiac output higher (p < 0.001) in LVAD patients; V30(ex vivo ventricular volume yielding 30 mm Hg, an index of ventricular size) in LVAD patients was decreased in the LV (p < 0.05) but did not change significantly in RV. The LVAD support improved force generation in response to beta-adrenergic stimulation in isolated LV (increase in developed force from 6.3 ± 0.6 to 18.5 ± 4.4 mN/m2, p < 0.01) and RV (increase in developed force, from 10.9 ± 2.0 to 20.5 ± 3.1 mN/m2, p < 0.05) trabeculae. The LVAD patients had higher myocardial beta-adrenergic receptor density in LV (p < 0.01) and RV (p < 0.01). Protein kinase A (PKA) hyperphosphorylation of the ryanodine receptor 2 (RyR2)/calcium release channel was significantly reduced by LVAD in both RV and LV (p < 0.01).ConclusionsImproved beta-adrenergic responsiveness, normalization of the RyR2 PKA phosphorylation, and increased beta-adrenergic receptor density in LV and RV after LVAD support suggest a primary role of neurohormonal environment in determining reverse remodeling of the beta-adrenergic pathway.

The cardiac slow delayed rectifier potassium channel (IKs), comprised of α (KCNQ1) and β (KCNE1) subunits, is regulated by sympathetic nervous stimulation, with activation of β-adrenergic receptors PKA phosphorylating IKs channels. We examined the effects of β2-adrenergic receptors (β2-AR) on IKs in cardiac ventricular myocytes from transgenic mice expressing fusion proteins of IKs subunits and hβ2-ARs. KCNQ1 and β2-ARs were localized to the same subcellular regions, sharing intimate localization within nanometers of each other. In IKs/B2-AR myocytes, IKs density was increased, and activation shifted in the hyperpolarizing direction; IKs was not further modulated by exposure to isoproterenol, and KCNQ1 was found to be PKA-phosphorylated. Conversely, β2-AR overexpression did not affect L-type calcium channel current (ICaL) under basal conditions with ICaL remaining responsive to cAMP. These data indicate intimate association of KCNQ1 and β2-ARs and that β2-AR signaling can modulate the function of IKs channels under conditions of increased β2-AR expression, even in the absence of exogenous β-AR agonist.

Arrhythmias, a common cause of sudden cardiac death, can occur in structurally normal hearts, although the mechanism is not known. In cardiac muscle, the ryanodine receptor (RyR2) on the sarcoplasmic reticulum releases the calcium required for muscle contraction. The FK506 binding protein (FKBP12.6) stabilizes RyR2, preventing aberrant activation of the channel during the resting phase of the cardiac cycle. We show that during exercise, RyR2 phosphorylation by cAMP-dependent protein kinase A (PKA) partially dissociates FKBP12.6 from the channel, increasing intracellular Ca 2+ release and cardiac contractility. FKBP12.6 −/− mice consistently exhibited exercise-induced cardiac ventricular arrhythmias that cause sudden cardiac death. Mutations in RyR2 linked to exercise-induced arrhythmias (in patients with catecholaminergic polymorphic ventricular tachycardia [CPVT]) reduced the affinity of FKBP12.6 for RyR2 and increased single-channel activity under conditions that simulate exercise. These data suggest that "leaky" RyR2 channels can trigger fatal cardiac arrhythmias, providing a possible explanation for CPVT.

The cardiac ryanodine receptor/calcium release channel (RyR2) on the sarcoplasmic reticulum (SR) comprises a macromolecular complex that includes a kinase and two phosphatases that are bound to the channel via targeting proteins. We previously found that the RyR2 is protein kinase A (PKA)-hyperphosphorylated in end-stage human heart failure. Because heart failure is a progressive disease that often evolves from hypertrophy, we analyzed the RyR2 macromolecular complex in several animal models of cardiomyopathy that lead to heart failure, including hypertrophy, and at different stages of disease progression. We now show that RyR2 is PKA-hyperphosphorylated in diverse models of heart failure and that the degree of RyR2 PKA phosphorylation correlates with the degree of cardiac dysfunction. Interestingly, we show that RyR2 PKA hyperphosphorylation can be lost during perfusion of isolated hearts due to the activity of the endogenous phosphatases in the RyR2 macromolecular complex. Moreover, infusion of isoproterenol resulted in PKA phosphorylation of RyR2 in rat, indicating that systemic catecholamines can activate phosphorylation of RyR2 in vivo. These studies extend our previous analyses of the RyR2 macromolecular complex, show that both the kinase and phosphatase activities in the macromolecular complex are regulated physiologically in vivo, and suggest that RyR2 PKA hyperphosphorylation is likely a general feature of heart failure.

Defective calcium (Ca(2+)) signaling, manifest as a loss of excitation-contraction (EC) coupling gain in cardiac muscle, likely plays an important role in the pathophysiology of human heart failure. The mechanism underlying this loss of cardiac EC coupling gain involves altered regulation of the cardiac ryanodine receptor (RyR2), the major sarcoplasmic reticulum Ca(2+) release channel in the heart. This altered regulation of RyR2 is due, in part, to hyperphosphorylation of the channel by cyclic adenosine monophosphate-dependent protein kinase A (PKA). PKA phosphorylation of RyR2 is controlled by a macromolecular signaling complex that targets PKA and two phosphatases (PP1 and PP2A) to the channel. The targeting of PKA, PP1, and PP2A to RyR2 is dependent on the binding of targeting proteins to the channel via highly conserved leucine/isoleucine zippers (LIZs). Formation of an ion channel macromolecular signaling complex is a novel role of LIZs. Recognition of this new function for LIZ motifs has provided a road map for rapidly identifying components of the RyR2 macromolecular signaling complex that play a key role in regulating normal cardiac physiology as part of the "fight or flight" response. The components of the RyR2 macromolecular signaling complex are also novel targets for heart failure and cardiac arrhythmia therapeutics.

The ryanodine receptor (RyR) is a conserved, calcium release channel located in the endo/sarcoplasmic reticulum (ER/SR). RyR1 is the predominant isoform in skeletal muscle and a crucial mediator of excitation-contraction coupling. It comprises a macromolecular complex that includes the channel stabilizing protein calstabin1. Posttranslational modification of RyR1 results in calstabin1 depletion and intracellular calcium leak. In aged skeletal muscle, this leak is hypothesized to reduce the membrane potential of nearby mitochondria, which subsequently overproduce reactive oxygen species (ROS) and further oxidize RyR1, exacerbating calcium leak/muscle dysfunction. The nematode C. elegans is a well-established model with a short lifespan, ideal for aging studies; however, the underlying physiology of muscle dysfunction in C. elegans aging and the role of ROS are unknown. Therefore, we have characterized the role of calcium leak in age-dependent decline of C. elegans muscle function by examining the oxidation state of its RyR homologue, UNC-68, in WT and FKB-2 (calstabin1 homologue) KO worm strains. Our data demonstrate that UNC-68 is a macromolecular complex highly homologous to RyR1; in WT aged worms, UNC-68 is oxidized and FKB-2 is depleted from the channel. Furthermore, UNC-68 becomes oxidized earlier in the FKB-2 KO worm lifespan. FKB-2 depletion reduces peak calcium beforeand after caffeine-induced UNC-68 activation. Finally, FKB-2 KO worms have decreased motility following swimming exertion. Further studies will include ROS measurements via mitochondrial targeted, redox sensitive GFP and lifespan assays. Our goal is to demonstrate intracellular calcium leak's importance in muscle pathophysiology and establish C. elegans as a tractable model of calcium signaling mechanisms therein.

The ryanodine receptor (RyR)/calcium release channel on the sarcoplasmic reticulum (SR) is the major source of calcium (Ca2+) required for cardiac muscle excitation-contraction (EC) coupling. The channel is a tetramer comprised of four type 2 RyR polypeptides (RyR2) and four FK506 binding proteins (FKBP12.6). We show that protein kinase A (PKA) phosphorylation of RyR2 dissociates FKBP12.6 and regulates the channel open probability (Po). Using cosedimentation and coimmunoprecipitation we have defined a macromolecular complex comprised of RyR2, FKBP12.6, PKA, the protein phosphatases PP1 and PP2A, and an anchoring protein, mAKAP. In failing human hearts, RyR2 is PKA hyperphosphorylated, resulting in defective channel function due to increased sensitivity to Ca2+-induced activation.

Les arythmies ventriculaires gauches representent une des principales causes de deces chez les patients atteint de la Dystrophie Musculaire de Duchenne (DMD). Dans le muscle squelettique, nous avons mis evidence une augmentation de la s-nytrosylation du canal calcique de type 1 du reticulum sarcoplasmique (RS), le recepteur a la ryanodine (RyR1), a l’origine d’une augmentation de la fuite spontanee du Ca2+ du RS (Bellinger et al (2009), Nature Med,). L’objectif de ce travail, a ete de determiner si une s-nytrolsylation accrue de l’isoforme cardiaque du RyR (RyR2) pouvait rendre compte des arythmies ventriculaires chez des souris deficientes en dystrophine, les souris mdx. En western blot, l’augmentation de la s-nytrosylation du RyR2 dans les cœurs de souris mdx apparait des l’ages de 40 jours. Cette s-nytrosylation est associee a la dissociation de la sous-unite regulatrice du RyR2, la calstabin 2 (ou FKBP12.6), et est a l’origine d’une augmentation de la fuite spontanee du Ca2+ du RS, mesuree par microscopie confocale (Fluo-4) sur des cardiomyocytes ventriculaires isoles. 10 jours de traitement par voie orale avec le S107 (Rycal) qui stabilise la liaison de la calstabin 2 avec le RyR2, empeche la fuite du Ca2+ du RS, normalise la signalisation Ca2+, et previent le declenchement d’arythmies induit par une stimulation adrenergique que ce soit au niveau cellulaire (Isoprenaline : 100 nM) ou in vivo (Isoprenaline : 2 mg/kg). En conclusion, des les premiers stades de la pathologie, la s-nytrosylation du RyR2 induit la dissociation de la calstabin 2 du RyR2 et augmente la fuite diastolique de Ca2+ ce qui dans des conditions de stress augmente la prevalence des arythmies. Une normalisation precoce de la fonction du RyR2 apparait comme une cible therapeutique nouvelle dans la prevention des phenomenes arythmogenes au cours de la DMD.

A hallmark of mammalian aging is the progressive decline in muscle function, referred to as sarcopenia. It is commonly found that the force-generating capacity of aged muscle is reduced when normalized to the muscle cross-sectional area (specific force). This indicates defective excitation-contraction coupling. In this process, sarcoplasmic reticulum (SR) Ca2+ release via the ryanodine receptor 1 (RyR1) is a pivotal step that grades muscle contractile force. It has previously been shown that impaired contractility and SR Ca2+ release in muscular dystrophy can be caused by excessive RyR1-cysteine nitrosylation and reduced binding of the stabilizing protein FK506 binding protein 12 (FKBP12 or calstabin1) to RyR1. We hypothesized that maladaptations in the RyR1-SR Ca2+ release system could underlie impaired muscle function also in aging. Using immunoprecipitation and immuonblotting, we found that RyR1 from aged (24-26 month) mouse muscle were oxidized, cysteine-nitrosylated, and depleted of FKBP12, compared to RyR1 from younger (3-6 months) adult mice. This remodeling of the RyR1 resulted in “leaky” channels, which displayed an increased open probability and Ca2+ spark frequency.Moreover, tetanic Ca2+ transients and muscle specific forcewere reduced in 24-month-old mice. Treating aged mice with the RyR-stabilizing compound, S107, restored RyR1-FKBP12 interaction, and improved tetanic Ca2+ release, muscle specific force and exercise capacity.Together, these findings highlight the role of impaired SR Ca2+ release in age-dependent muscle weakness and introduce a novel therapeutic target for sarcopenia.

Activation of the type 1 ryanodine receptor (RyR1) during skeletal muscle excitation-contraction (EC) coupling allows the release of stored Ca2+ required for muscle contraction. While RyR1 is a well-known substrate of protein kinase A (PKA), the physiological significance of this phosphorylation event is poorly defined. PKA is known to increase the open probability of the channel in lipid bilayers by inducing phosphorylation of a specific residue (S2844). Knock-in mice harboring phosphorylation site deficient (RyR1-S2844A) and phosphomimetic (RyR1-S2844D) mutations were generated in order to probe the consequence of this phosphorylation event in skeletal muscle EC coupling. Specific force measurements were performed on extensor digitorum longus (EDL) muscles and intracellular Ca2+ release was examined in isolated flexor digitorum brevis (FDB) muscle fibers using confocal microscopy. While isoproterenol (ISO) enhanced both Ca2+ release and muscle force in wild type (WT) mice, the positive effects of ISO were abrogated in samples from RyR1-S2844A mice. This demonstrates the enhancing effects of RyR1 phosphorylation during adrenergic stimulation in skeletal muscle. While transient phosphorylation of RyR1 exerts positive effects, chronic phosphorylation, as occurs in animal heart failure models, is thought to be detrimental to muscle function. We tested this hypothesis by measuring muscle force production and intracellular Ca2+ in RyR1-S2844D mice. At 3-months of age, muscle function and Ca2+ release were similar to WT controls. However, muscle force production and intracellular Ca2+ release were both blunted by 6-months of age suggesting that a pathological Ca2+ leak induced by the S2844D mutation impairs muscle function.