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Mutations in the lamin A/C gene (LMNA), which encodes A-type lamins, cause several diseases called laminopathies, the most common of which is dilated cardiomyopathy with muscular dystrophy. The role of Ca2+ regulation in these diseases remain poorly understood. We now show biochemical remodeling of the ryanodine receptor (RyR)/intracellular Ca2+ release channel in heart samples from human subjects with LMNA mutations, including protein kinase A-catalyzed phosphorylation, oxidation and depletion of the stabilizing subunit calstabin. In the LmnaH222P/H222P murine model of Emery-Dreifuss muscular dystrophy caused by LMNA mutation, we demonstrate an age-dependent biochemical remodeling of RyR2 in the heart and RyR1 in skeletal muscle. This RyR remodeling is associated with heart and skeletal muscle dysfunction. Defective heart and muscle function are ameliorated by treatment with a novel Rycal small molecule drug (S107) that fixes ‘leaky’ RyRs. SMAD3 phosphorylation is increased in hearts and diaphragms of LmnaH222P/H222P mice, which enhances NADPH oxidase binding to RyR channels, contributing to their oxidation. There is also increased generalized protein oxidation, increased calcium/calmodulin-dependent protein kinase II-catalyzed phosphorylation of RyRs and increased protein kinase A activity in these tissues. Our data show that RyR remodeling plays a role in cardiomyopathy and skeletal muscle dysfunction caused by LMNA mutation and identify these Ca2+ channels as a potential therapeutic target.

International audience; Objectives: Mechanical ventilation is associated with primary diaphragmatic dysfunction, also termed ventilator-induced diaphragmatic dysfunction. Studies evaluating diaphragmatic function recovery after extubation are lacking. We evaluated early and late recoveries from ventilator-induced diaphragmatic dysfunction in a mouse model.Design: Experimental randomized study.Setting: Research laboratory.Subjects: C57/BL6 mice.Interventions: Six groups of C57/BL6 mice. Mice were ventilated for 6 hours and then euthanatized immediately (n = 18), or 1 (n = 18) or 10 days after extubation with (n = 5) and without S107 (n = 16) treatment. Mice euthanatized immediately after 6 hours of anesthesia (n = 15) or after 6 hours of anesthesia and 10 days of recovery (n = 5) served as controls.Measurements and Main Results: For each group, diaphragm force production, posttranslational modification of ryanodine receptor, oxidative stress, proteolysis, and cross-sectional areas were evaluated. After 6 hours of mechanical ventilation, diaphragm force production was decreased by 25–30%, restored to the control levels 1 day after extubation, and secondarily decreased by 20% 10 days after extubation compared with controls. Ryanodine receptor was protein kinase A-hyperphosphorylated, S-nitrosylated, oxidized, and depleted of its stabilizing subunit calstabin-1 6 hours after the onset of the mechanical ventilation, 1 and 10 days after extubation. Post extubation treatment with S107, a Rycal drug that stabilizes the ryanodine complex, did reverse the loss of diaphragmatic force associated with mechanical ventilation. Total protein oxidation was restored to the control levels 1 day after extubation. Markers of proteolysis including calpain 1 and calpain 2 remained activated 10 days after extubation without significant changes in cross-sectional areas.Conclusions: We report that mechanical ventilation is associated with a late diaphragmatic dysfunction related to a structural alteration of the ryanodine complex that is reversed with the S107 treatment.

The mechanisms underlying ryanodine receptor (RyR) dysfunction associated with Alzheimer disease (AD) are still not well understood. Here, we show that neuronal RyR2 channels undergo post-translational remodeling (PKA phosphorylation, oxidation, and nitrosylation) in brains of AD patients, and in two murine models of AD (3 × Tg-AD, APP +/− /PS1 +/−). RyR2 is depleted of calstabin2 (KFBP12.6) in the channel complex, resulting in endoplasmic reticular (ER) calcium (Ca2+) leak. RyR-mediated ER Ca2+ leak activates Ca2+-dependent signaling pathways, contributing to AD pathogenesis. Pharmacological (using a novel RyR stabilizing drug Rycal) or genetic rescue of the RyR2-mediated intracellular Ca2+ leak improved synaptic plasticity, normalized behavioral and cognitive functions and reduced Aβ load. Genetically altered mice with congenitally leaky RyR2 exhibited premature and severe defects in synaptic plasticity, behavior and cognitive function. These data provide a mechanism underlying leaky RyR2 channels, which could be considered as potential AD therapeutic targets.

The type 2 ryanodine receptor (RyR2) is a Ca2+ release channel on the endoplasmic reticulum (ER) of several types of cells, including cardiomyocytes and pancreatic beta cells. In cardiomyocytes, RyR2-dependent Ca2+ release is critical for excitation-contraction coupling; however, a functional role for RyR2 in beta cell insulin secretion and diabetes mellitus remains controversial. Here, we took advantage of rare RyR2 mutations that were identified in patients with a genetic form of exercise-induced sudden death (catecholaminergic polymorphic ventricular tachycardia [CPVT]). As these mutations result in a "leaky" RyR2 channel, we exploited them to assess RyR2 channel function in p cell dynamics. We discovered that CPVT patients with mutant leaky RyR2 present with glucose intolerance, which was heretofore unappreciated. In mice, transgenic expression of CPVT-associated RyR2 resulted in impaired glucose homeostasis, and an in-depth evaluation of pancreatic islets and p cells from these animals revealed intracellular Ca2+ leak via oxidized and nitrosylated RyR2 channels, activated ER stress response, mitochondrial dysfunction, and decreased fuel-stimulated insulin release. Additionally, we verified the effects of the pharmacological inhibition of intracellular Ca2+ leak in CPVT-associated RyR2-expressing mice, in human islets from diabetic patients, and in an established murine model of type 2 diabetes mellitus. Taken together, our data indicate that RyR2 channels play a crucial role in the regulation of insulin secretion and glucose homeostasis.

Muscle weakness and cachexia are significant paraneoplastic syndromes of many advanced cancers. Osteolytic bone metastases are common in advanced breast cancer and are a major contributor to decreased survival, performance, and quality of life for patients. Pathologic fracture caused by osteolytic cancer in bone (OCIB) leads to a significant (32%) increased risk of death compared to patients without fracture. Since muscle weakness is linked to risk of falls which are a major cause of fracture, we have investigated skeletal muscle response to OCIB. Here, we show that a syngeneic mouse model of OCIB (4T1 mammary tumor cells) leads to cachexia and skeletal muscle weakness associated with oxidation of the ryanodine receptor and calcium (Ca2+) release channel (RyR1). Muscle atrophy follows known pathways via both myostatin signaling and expression of muscle-specific ubiquitin ligases, atrogin-1 and MuRF1. We have identified a mechanism for skeletal muscle weakness due to increased oxidative stress on RyR1 via NAPDH oxidases [NADPH oxidase 2 (Nox2) and NADPH oxidase 4 (Nox4)]. In addition, SMAD3 phosphorylation is higher in muscle from tumor-bearing mice, a critical step in the intracellular signaling pathway that transmits TGFβ signaling to the nucleus. This is the first time that skeletal muscle weakness has been described in a syngeneic model of OCIB and represents a unique model system in which to study cachexia and changes in skeletal muscle.

Ryanodine receptors (RyRs) mediate the rapid release of calcium (Ca2+) from intracellular stores into the cytosol, which is essential for numerous cellular functions including excitation–contraction coupling in muscle. Lack of sufficient structural detail has impeded understanding of RyR gating and regulation. Here we report the closed-state structure of the 2.3-megadalton complex of the rabbit skeletal muscle type 1 RyR (RyR1), solved by single-particle electron cryomicroscopy at an overall resolution of 4.8 A. We fitted a polyalanine-level model to all 3,757 ordered residues in each protomer, defining the transmembrane pore in unprecedented detail and placing all cytosolic domains as tertiary folds. The cytosolic assembly is built on an extended α-solenoid scaffold connecting key regulatory domains to the pore. The RyR1 pore architecture places it in the six-transmembrane ion channel superfamily. A unique domain inserted between the second and third transmembrane helices interacts intimately with paired EF-hands originating from the α-solenoid scaffold, suggesting a mechanism for channel gating by Ca2+. Using electron cryomicroscopy, the closed-state structure of rabbit RyR1 is determined at 4.8 A resolution; analysis confirms that the RyR1 architecture consists of a six-transmembrane ion channel with a cytosolic α-solenoid scaffold, and suggests a mechanism for Ca2+-induced channel opening. Muscle contraction is regulated by the concentration of calcium ions in the cytoplasm of muscle cells. Ryanodine receptors (RyR) release Ca2+ from the sarcoplasmic reticulum to induce muscle contraction. Dysfunction of these channels contributes to the pathophysiology of important human diseases including muscular dystrophy. Three papers in this issue of Nature report high-resolution electron cryomicroscopy structures of the 2.2 MDa ryanodine receptor RyR1. Efremov et al. report the structure of rabbit RyR1 at 8.5 A resolution the presence of Ca2+ in a 'partly open' state, and at 6.1 A resolution in the absence of Ca2+ in a closed state. Zalk et al. report the rabbit RyR1 structure at 4.8 A in the absence of Ca2+ in a closed state. And third, Yan et al. report the structure of rabbit RyR1 bound to its modulator FKBP12 at a near-atomic resolution of 3.8 A. These papers reveal how calcium binding to the EF-hand domain of RyR1 regulates channel opening and facilitates calcium-induced calcium release. The authors also note that disease-causing mutations are clustered in regions of the channel that appear to be critical for normal channel function.

Ventilator-induced diaphragmatic dysfunction (VIDD) refers to the diaphragm muscle weakness that occurs following prolonged controlled mechanical ventilation (MV). The presence of VIDD impedes recovery from respiratory failure. However, the pathophysiological mechanisms accounting for VIDD are still not fully understood. Here, we show in human subjects and a mouse model of VIDD that MV is associated with rapid remodeling of the sarcoplasmic reticulum (SR) Ca(2+) release channel/ryanodine receptor (RyR1) in the diaphragm. The RyR1 macromolecular complex was oxidized, S-nitrosylated, Ser-2844 phosphorylated, and depleted of the stabilizing subunit calstabin1, following MV. These posttranslational modifications of RyR1 were mediated by both oxidative stress mediated by MV and stimulation of adrenergic signaling resulting from the anesthesia. We demonstrate in the murine model that such abnormal resting SR Ca(2+) leak resulted in reduced contractile function and muscle fiber atrophy for longer duration of MV. Treatment with β-adrenergic antagonists or with S107, a small molecule drug that stabilizes the RyR1-calstabin1 interaction, prevented VIDD. Diaphragmatic dysfunction is common in MV patients and is a major cause of failure to wean patients from ventilator support. This study provides the first evidence to our knowledge of RyR1 alterations as a proximal mechanism underlying VIDD (i.e., loss of function, muscle atrophy) and identifies RyR1 as a potential target for therapeutic intervention.

International audience; Endothelial cells (ECs) are critical mediators of blood pressure (BP) regulation, primarily via the generation and release of vasorelaxants, including nitric oxide (NO). NO is produced in ECs by endothelial NO synthase (eNOS), which is activated by both calcium (Ca(2+))-dependent and independent pathways. Here, we report that intracellular Ca(2+) release from the endoplasmic reticulum (ER) via inositol 1,4,5-trisphosphate receptor (IP3R) is required for Ca(2+)-dependent eNOS activation. EC-specific type 1 1,4,5-trisphosphate receptor knockout (IP3R1(-/-)) mice are hypertensive and display blunted vasodilation in response to acetylcholine (ACh). Moreover, eNOS activity is reduced in both isolated IP3R1-deficient murine ECs and human ECs following IP3R1 knockdown. IP3R1 is upstream of calcineurin, a Ca(2+)/calmodulin-activated serine/threonine protein phosphatase. We show here that the calcineurin/nuclear factor of activated T cells (NFAT) pathway is less active and eNOS levels are decreased in IP3R1-deficient ECs. Furthermore, the calcineurin inhibitor cyclosporin A, whose use has been associated with the development of hypertension, reduces eNOS activity and vasodilation following ACh stimulation. Our results demonstrate that IP3R1 plays a crucial role in the EC-mediated vasorelaxation and the maintenance of normal BP.

Enhancement of contractile force (inotropy) occurs in skeletal muscle following neuroendocrine release of catecholamines and activation of muscle β-adrenergic receptors. Despite extensive study, the molecular mechanism underlying the inotropic response in skeletal muscle is not well understood. Here we show that phosphorylation of a single serine residue (S2844) in the sarcoplasmic reticulum (SR) Ca(2+) release channel/ryanodine receptor type 1 (RyR1) by protein kinase A (PKA) is critical for skeletal muscle inotropy. Treating fast twitch skeletal muscle from wild-type mice with the β-receptor agonist isoproterenol (isoprenaline) increased RyR1 PKA phosphorylation, twitch Ca(2+) and force generation. In contrast, the enhanced muscle Ca(2+), force and in vivo muscle strength responses following isoproterenol stimulation were abrogated in RyR1-S2844A mice in which the serine in the PKA site in RyR1 was replaced with alanine. These data suggest that the molecular mechanism underlying skeletal muscle inotropy requires enhanced SR Ca(2+) release due to PKA phosphorylation of S2844 in RyR1.

Rationale: Atrial fibrillation (AF) is the most common cardiac arrhythmia, however the mechanism(s) causing AF remain poorly understood and therapy is suboptimal. The ryanodine receptor (RyR2) is the major calcium (Ca 2+ ) release channel on the sarcoplasmic reticulum (SR) required for excitation-contraction coupling in cardiac muscle. Objective: In the present study, we sought to determine whether intracellular diastolic SR Ca 2+ leak via RyR2 plays a role in triggering AF and whether inhibiting this leak can prevent AF. Methods and Results: We generated 3 knock-in mice with mutations introduced into RyR2 that result in leaky channels and cause exercise induced polymorphic ventricular tachycardia in humans [catecholaminergic polymorphic ventricular tachycardia (CPVT)]. We examined AF susceptibility in these three CPVT mouse models harboring RyR2 mutations to explore the role of diastolic SR Ca 2+ leak in AF. AF was stimulated with an intra-esophageal burst pacing protocol in the 3 CPVT mouse models (RyR2-R2474S +/− , 70%; RyR2-N2386I +/− , 60%; RyR2-L433P +/− , 35.71%) but not in wild-type (WT) mice ( P 2+ leak in atrial myocytes isolated from the CPVT mouse models. Calstabin2 (FKBP12.6) is an RyR2 subunit that stabilizes the closed state of RyR2 and prevents a Ca 2+ leak through the channel. Atrial RyR2 from RyR2-R2474S +/− mice were oxidized, and the RyR2 macromolecular complex was depleted of calstabin2. The Rycal drug S107 stabilizes the closed state of RyR2 by inhibiting the oxidation/phosphorylation induced dissociation of calstabin2 from the channel. S107 reduced the diastolic SR Ca 2+ leak in atrial myocytes and decreased burst pacing–induced AF in vivo. S107 did not reduce the increased prevalence of burst pacing–induced AF in calstabin2-deficient mice, confirming that calstabin2 is required for the mechanism of action of the drug. Conclusions: The present study demonstrates that RyR2-mediated diastolic SR Ca 2+ leak in atrial myocytes is associated with AF in CPVT mice. Moreover, the Rycal S107 inhibited diastolic SR Ca 2+ leak through RyR2 and pacing-induced AF associated with CPVT mutations.

Antagonists of L-type Ca²⁺ channels (LTCCs) have been used to treat human cardiovascular diseases for decades. However, these inhibitors can have untoward effects in patients with heart failure, and their overall therapeutic profile remains nebulous given differential effects in the vasculature when compared with those in cardiomyocytes. To investigate this issue, we examined mice heterozygous for the gene encoding the pore-forming subunit of LTCC (calcium channel, voltage-dependent, L type, α1C subunit [Cacna1c mice; referred to herein as α1C⁻/⁺ mice]) and mice in which this gene was loxP targeted to achieve graded heart-specific gene deletion (termed herein α1C-loxP mice). Adult cardiomyocytes from the hearts of α1C⁻/⁺ mice at 10 weeks of age showed a decrease in LTCC current and a modest decrease in cardiac function, which we initially hypothesized would be cardioprotective. However, α1C⁻/⁺ mice subjected to pressure overload stimulation, isoproterenol infusion, and swimming showed greater cardiac hypertrophy, greater reductions in ventricular performance, and greater ventricular dilation than α1C⁺/⁺ controls. The same detrimental effects were observed in α1C-loxP animals with a cardiomyocyte-specific deletion of one allele. More severe reductions in α1C protein levels with combinatorial deleted alleles produced spontaneous cardiac hypertrophy before 3 months of age, with early adulthood lethality. Mechanistically, our data suggest that a reduction in LTCC current leads to neuroendocrine stress, with sensitized and leaky sarcoplasmic reticulum Ca²⁺ release as a compensatory mechanism to preserve contractility. This state results in calcineurin/nuclear factor of activated T cells signaling that promotes hypertrophy and disease.

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.

The force frequency relationship (FFR), first described by Bowditch 139 years ago as the observation that myocardial contractility increases proportionally with increasing heart rate, is an important mediator of enhanced cardiac output during exercise. Individuals with heart failure have defective positive FFR that impairs their cardiac function in response to stress, and the degree of positive FFR deficiency correlates with heart failure progression. We have identified a mechanism for FFR involving heart rate dependent phosphorylation of the major cardiac sarcoplasmic reticulum calcium release channel/ryanodine receptor (RyR2), at Ser2814, by calcium/calmodulin-dependent serine/threonine kinase-delta (CaMKIIdelta). Mice engineered with an RyR2-S2814A mutation have RyR2 channels that cannot be phosphorylated by CaMKIIdelta, and exhibit a blunted positive FFR. Ex vivo hearts from RyR2-S2814A mice also have blunted positive FFR, and cardiomyocytes isolated from the RyR2-S2814A mice exhibit impaired rate-dependent enhancement of cytosolic calcium levels and fractional shortening. The cardiac RyR2 macromolecular complexes isolated from murine and human failing hearts have reduced CaMKIIdelta levels. These data indicate that CaMKIIdelta phosphorylation of RyR2 plays an important role in mediating positive FFR in the heart, and that defective regulation of RyR2 by CaMKIIdelta-mediated phosphorylation is associated with the loss of positive FFR in failing hearts.

During exercise, defects in calcium (Ca 2+ ) release have been proposed to impair muscle function. Here, we show that during exercise in mice and humans, the major Ca 2+ release channel required for excitation–contraction coupling (ECC) in skeletal muscle, the ryanodine receptor (RyR1), is progressively PKA-hyperphosphorylated, S -nitrosylated, and depleted of the phosphodiesterase PDE4D3 and the RyR1 stabilizing subunit calstabin1 (FKBP12), resulting in “leaky” channels that cause decreased exercise tolerance in mice. Mice with skeletal muscle-specific calstabin1 deletion or PDE4D deficiency exhibited significantly impaired exercise capacity. A small molecule (S107) that prevents depletion of calstabin1 from the RyR1 complex improved force generation and exercise capacity, reduced Ca 2+ -dependent neutral protease calpain activity and plasma creatine kinase levels. Taken together, these data suggest a possible mechanism by which Ca 2+ leak via calstabin1-depleted RyR1 channels leads to defective Ca 2+ signaling, muscle damage, and impaired exercise capacity.

Oxidative stress has been suggested to play a role in the pathogenesis of atrial fibrillation (AF). Indeed, the prevalence of AF increases with age as does oxidative stress. However, the mechanisms linking redox state to AF are not well understood. In this study we identify a link between oxidative stress and aberrant intracellular Ca2+ release via the type 2 ryanodine receptor (RyR2) that promotes AF. We show that RyR2 are oxidized in the atria of patients with chronic AF compared with individuals in sinus rhythm. To dissect the molecular mechanism linking RyR2 oxidation to AF we used two murine models harboring RyR2 mutations that cause intracellular Ca2+ leak. Mice with intracellular Ca2+ leak exhibited increased atrial RyR2 oxidation, mitochondrial dysfunction, reactive oxygen species (ROS) production and AF susceptibility. Both genetic inhibition of mitochondrial ROS production and pharmacological treatment of RyR2 leakage prevented AF. Collectively, our results indicate that alterations of RyR2 and mitochondrial ROS generation form a vicious cycle in the development of AF. Targeting this previously unrecognized mechanism could be useful in developing effective interventions to prevent and treat AF.

Cancer-associated muscle weakness is a poorly understood phenomenon, and there is no effective treatment. Here we find that seven different mouse models of human osteolytic bone metastases-representing breast, lung and prostate cancers, as well as multiple myeloma-exhibited impaired muscle function, implicating a role for the tumor-bone microenvironment in cancer-associated muscle weakness. We found that transforming growth factor (TGF)-β, released from the bone surface as a result of metastasis-induced bone destruction, upregulated NADPH oxidase 4 (Nox4), resulting in elevated oxidization of skeletal muscle proteins, including the ryanodine receptor and calcium (Ca(2+)) release channel (RyR1). The oxidized RyR1 channels leaked Ca(2+), resulting in lower intracellular signaling, which is required for proper muscle contraction. We found that inhibiting RyR1 leakage, TGF-β signaling, TGF-β release from bone or Nox4 activity improved muscle function in mice with MDA-MB-231 bone metastases. Humans with breast- or lung cancer-associated bone metastases also had oxidized skeletal muscle RyR1 that is not seen in normal muscle. Similarly, skeletal muscle weakness, increased Nox4 binding to RyR1 and oxidation of RyR1 were present in a mouse model of Camurati-Engelmann disease, a nonmalignant metabolic bone disorder associated with increased TGF-β activity. Thus, pathological TGF-β release from bone contributes to muscle weakness by decreasing Ca(2+)-induced muscle force production.

Defective regulation of the cardiac ryanodine receptor (RyR2)/calcium release channel, required for excitation-contraction coupling in the heart, has been linked to cardiac arrhythmias and heart failure. For example, diastolic calcium “leak” via RyR2 channels in the sarcoplasmic reticulum has been identified as an important factor contributing to impaired contractility in heart failure and ventricular arrhythmias that cause sudden cardiac death. In patients with heart failure, chronic activation of the “fight or flight” stress response leads to protein kinase A (PKA) hyperphosphorylation of RyR2 at Ser-2808. PKA phosphorylation of RyR2 Ser-2808 reduces the binding affinity of the channel-stabilizing subunit calstabin2, resulting in leaky RyR2 channels. We developed RyR2-S2808A mice to determine whether Ser-2808 is the functional PKA phosphorylation site on RyR2. Furthermore, mice in which the RyR2 channel cannot be PKA phosphorylated were relatively protected against the development of heart failure after myocardial infarction. Taken together, these data show that PKA phosphorylation of Ser-2808 on the RyR2 channel appears to be a critical mediator of progressive cardiac dysfunction after myocardial infarction.

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.

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 type 1 ryanodine receptor (RyR1) on the sarcoplasmic reticulum (SR) is the major calcium (Ca2+) release channel required for skeletal muscle excitation–contraction (EC) coupling. RyR1 function is modulated by proteins that bind to its large cytoplasmic scaffold domain, including the FK506 binding protein (FKBP12) and PKA. PKA is activated during sympathetic nervous system (SNS) stimulation. We show that PKA phosphorylation of RyR1 at Ser2843 activates the channel by releasing FKBP12. When FKB12 is bound to RyR1, it inhibits the channel by stabilizing its closed state. RyR1 in skeletal muscle from animals with heart failure (HF), a chronic hyperadrenergic state, were PKA hyperphosphorylated, depleted of FKBP12, and exhibited increased activity, suggesting that the channels are “leaky.” RyR1 PKA hyperphosphorylation correlated with impaired SR Ca2+ release and early fatigue in HF skeletal muscle. These findings identify a novel mechanism that regulates RyR1 function via PKA phosphorylation in response to SNS stimulation. PKA hyperphosphorylation of RyR1 may contribute to impaired skeletal muscle function in HF, suggesting that a generalized EC coupling myopathy may play a role in HF.

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.

Calcium (Ca2+) released from the sarcoplasmic reticulum (SR) is crucial for excitation–contraction (E–C) coupling. Mitochondria, the major source of energy, in the form of ATP, required for cardiac contractility, are closely interconnected with the SR, and Ca2+ is essential for optimal function of these organelles. However, Ca2+ accumulation can impair mitochondrial function, leading to reduced ATP production and increased release of reactive oxygen species (ROS). Oxidative stress contributes to heart failure (HF), but whether mitochondrial Ca2+ plays a mechanistic role in HF remains unresolved. Here, we show for the first time, to our knowledge, that diastolic SR Ca2+ leak causes mitochondrial Ca2+ overload and dysfunction in a murine model of postmyocardial infarction HF. There are two forms of Ca2+ release channels on cardiac SR: type 2 ryanodine receptors (RyR2s) and type 2 inositol 1,4,5-trisphosphate receptors (IP3R2s). Using murine models harboring RyR2 mutations that either cause or inhibit SR Ca2+ leak, we found that leaky RyR2 channels result in mitochondrial Ca2+ overload, dysmorphology, and malfunction. In contrast, cardiac-specific deletion of IP3R2 had no major effect on mitochondrial fitness in HF. Moreover, genetic enhancement of mitochondrial antioxidant activity improved mitochondrial function and reduced posttranslational modifications of RyR2 macromolecular complex. Our data demonstrate that leaky RyR2, but not IP3R2, channels cause mitochondrial Ca2+ overload and dysfunction in HF.

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 cardiac ryanodine receptor (RyR2)/Ca2+ release channel on the sarcoplasmic reticulum (SR) is regulated by evolutionarily highly conserved signaling pathways that control excitation-contraction (EC) coupling in the heart. Phosphorylation of RyR2 by cAMP-dependent protein kinase (PKA) plays a key role in regulating the channel in response to stress via activation of the sympathetic nervous system (the “fight-or-flight response”).1 Maladaptive PKA hyperphosphorylation of RyR2 in failing hearts alters channel function, which may cause depletion of SR Ca2+ and diastolic release of SR Ca2+. This can initiate delayed afterdepolarizations that trigger ventricular arrhythmias.1 Mutations in RyR2 recently have been identified in patients with catecholaminergic induced sudden cardiac death (SCD).2–4⇓⇓ There may be a direct link between the PKA hyperphosphorylation of RyR2 that occurs during the progression of heart failure and fatal cardiac arrhythmias. Regulation of cardiac EC coupling by the release of Ca2+ from the SR via RyR2 in cardiomyocytes, known as Ca2+-induced Ca2+ release (CICR), has been appreciated for more than a decade.5,6⇓ Furthermore, it is well known that the amplitude of the Ca2+ transient generated by SR Ca2+ release determines contractile force in cardiomyocytes. The systems that regulate SR Ca2+ release include: (1) the triggers (predominantly Ca2+ influx through the voltage-gated Ca2+ channel on the plasma membrane); (2) the SR Ca2+ release channel or type 2 RyR2; and (3) the SR Ca2+ reuptake pump (SERCA2a) and its regulator phospholamban. These systems (trigger, release, and reuptake) are modulated by signaling pathways, including the β-adrenergic receptor (β-AR) signaling pathway (ie, phosphorylation by PKA). Activation of the sympathetic nervous system in response to stress results in elevation of cAMP levels and activation of PKA. Phosphorylation of RyR2 may not correlate directly …

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.

β-Adrenergic receptor (βAR) signaling, which elevates intracellular cAMP and enhances cardiac contractility, is severely impaired in the failing heart. Protein kinase A (PKA) is activated by cAMP, but the long-term physiological effect of PKA activation on cardiac function is unclear. To investigate the consequences of chronic cardiac PKA activation in the absence of upstream events associated with βAR signaling, we generated transgenic mice that expressed the catalytic subunit of PKA in the heart. These mice developed dilated cardiomyopathy with reduced cardiac contractility, arrhythmias, and susceptibility to sudden death. As seen in human heart failure, these abnormalities correlated with PKA-mediated hyperphosphorylation of the cardiac ryanodine receptor/Ca 2+ -release channel, which enhances Ca 2+ release from the sarcoplasmic reticulum, and phospholamban, which regulates the sarcoplasmic reticulum Ca 2+ -ATPase. These findings demonstrate a specific role for PKA in the pathogenesis of heart failure, independent of more proximal events in βAR signaling, and support the notion that PKA activity is involved in the adverse effects of chronic βAR signaling.

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.

Age-related skeletal muscle dysfunction is a leading cause of morbidity that affects up to half the population aged 80 or greater. Here we tested the effects of increased mitochondrial antioxidant activity on age-dependent skeletal muscle dysfunction using transgenic mice with targeted overexpression of the human catalase gene to mitochondria (MCat mice). Aged MCat mice exhibited improved voluntary exercise, increased skeletal muscle specific force and tetanic Ca(2+) transients, decreased intracellular Ca(2+) leak and increased sarcoplasmic reticulum (SR) Ca(2+) load compared with age-matched wild type (WT) littermates. Furthermore, ryanodine receptor 1 (the sarcoplasmic reticulum Ca(2+) release channel required for skeletal muscle contraction; RyR1) from aged MCat mice was less oxidized, depleted of the channel stabilizing subunit, calstabin1, and displayed increased single channel open probability (Po). Overall, these data indicate a direct role for mitochondrial free radi! cals in promoting the pathological intracellular Ca(2+) leak that underlies age-dependent loss of skeletal muscle function. This study harbors implications for the development of novel therapeutic strategies, including mitochondria-targeted antioxidants for treatment of mitochondrial myopathies and other healthspan-limiting disorders.

Recent studies indicate that members of the multidrug-resistance protein (MRP) family belonging to ATP binding cassette type C (ABCC) membrane proteins extrude cyclic nucleotides from various cell types. This study aimed to determine whether MRP proteins regulate cardiac cAMP homeostasis. Here, we demonstrate that MRP4 is the predominant isoform present at the plasma membrane of cardiacmyocytes and that it mediates the efflux of cAMP in these cells. MRP4-deficient mice displayed enhanced cardiac myocyte cAMP formation, contractility, and cardiac hypertrophy at 9 mo of age, an effect that was compensated transiently by increased phosphodiesterase expression at young age. These findings suggest that cAMP extrusion via MRP4 acts together with phosphodiesterases to control cAMP levels in cardiac myocytes.—Sassi, Y., Abi-Gerges, A., Fauconnier, J., Mougenot, N., Reiken, S., Haghighi, K., Kranias, E. G., Marks, A. R., Lacampagne, A., Engelhardt, S., Hatem, S. N., Lompre, A.-M., Hulot, J. S. Regulation of cAMP homeostasis by the efflux protein MRP4 in cardiac myocytes.

Increased sarcoplasmic reticulum (SR) Ca2+ leak via the cardiac ryanodine receptor/calcium release channel (RyR2) is thought to play a role in heart failure (HF) progression. Inhibition of this leak is an emerging therapeutic strategy. To explore the role of chronic PKA phosphorylation of RyR2 in HF pathogenesis and treatment, we generated a knockin mouse with aspartic acid replacing serine 2808 (mice are referred to herein as RyR2-S2808D+/+ mice). This mutation mimics constitutive PKA hyperphosphorylation of RyR2, which causes depletion of the stabilizing subunit FKBP12.6 (also known as calstabin2), resulting in leaky RyR2. RyR2-S2808D+/+ mice developed age-dependent cardiomyopathy, elevated RyR2 oxidation and nitrosylation, reduced SR Ca2+ store content, and increased diastolic SR Ca2+ leak. After myocardial infarction, RyR2-S2808D+/+ mice exhibited increased mortality compared with WT littermates. Treatment with S107, a 1,4-benzothiazepine derivative that stabilizes RyR2-calstabin2 interactions, inhibited the RyR2-mediated diastolic SR Ca2+ leak and reduced HF progression in WT and RyR2-S2808D+/+ mice. In contrast, β-adrenergic receptor blockers improved cardiac function in WT but not in RyR2-S2808D+/+ mice.Thus, chronic PKA hyperphosphorylation of RyR2 results in a diastolic leak that causes cardiac dysfunction. Reversing PKA hyperphosphorylation of RyR2 is an important mechanism underlying the therapeutic action of β-blocker therapy in HF.

During the classic “fight-or-flight” stress response, sympathetic nervous system activation leads to catecholamine release, which increases heart rate and contractility, resulting in enhanced cardiac output. Catecholamines bind to β-adrenergic receptors, causing cAMP generation and activation of PKA, which phosphorylates multiple targets in cardiac muscle, including the cardiac ryanodine receptor/calcium release channel (RyR2) required for muscle contraction. PKA phosphorylation of RyR2 enhances channel activity by sensitizing the channel to cytosolic calcium (Ca2+). Here, we found that mice harboring RyR2 channels that cannot be PKA phosphorylated (referred to herein as RyR2-S2808A+/+ mice) exhibited blunted heart rate and cardiac contractile responses to catecholamines (isoproterenol). The isoproterenol-induced enhancement of ventricular myocyte Ca2+ transients and fractional shortening (contraction) and the spontaneous beating rate of sinoatrial nodal cells were all blunted in RyR2-S2808A+/+ mice. The blunted cardiac response to catecholamines in RyR2-S2808A+/+ mice resulted in impaired exercise capacity. RyR2-S2808A+/+ mice were protected against chronic catecholaminergic-induced cardiac dysfunction. These studies identify what we believe to be new roles for PKA phosphorylation of RyR2 in both the heart rate and contractile responses to acute catecholaminergic stimulation. peerReviewed

The Ca2+ release channel ryanodine receptor 2 (RyR2) is required for excitation-contraction coupling in the heart and is also present in the brain. Mutations in RyR2 have been linked to exercise-induced sudden cardiac death (catecholaminergic polymorphic ventricular tachycardia [CPVT]). CPVT-associated RyR2 mutations result in “leaky” RyR2 channels due to the decreased binding of the calstabin2 (FKBP12.6) subunit, which stabilizes the closed state of the channel. We found that mice heterozygous for the R2474S mutation in Ryr2 (Ryr2-R2474S mice) exhibited spontaneous generalized tonic-clonic seizures (which occurred in the absence of cardiac arrhythmias), exercise-induced ventricular arrhythmias, and sudden cardiac death. Treatment with a novel RyR2-specific compound (S107) that enhances the binding of calstabin2 to the mutant Ryr2-R2474S channel inhibited the channel leak and prevented cardiac arrhythmias and raised the seizure threshold. Thus, CPVT-associated mutant leaky Ryr2-R2474S channels in the brain can cause seizures in mice, independent of cardiac arrhythmias. Based on these data, we propose that CPVT is a combined neurocardiac disorder in which leaky RyR2 channels in the brain cause epilepsy, and the same leaky channels in the heart cause exercise-induced sudden cardiac death.

When acutely exposed to a cold environment, mammals shiver to generate heat. During prolonged cold exposure, shivering is replaced by adaptive adrenergic nonshivering thermogenesis with increased heat production in brown adipose tissue due to activation of uncoupling protein-1 (UCP1). This cold acclimation is associated with chronically increased sympathetic stimulation of skeletal muscle, which may increase the sarcoplasmic reticulum (SR) Ca2+ leak via destabilized ryanodine receptor 1 (RyR1) channel complexes. Here, we use genetically engineered UCP1-deficient (UCP1-KO) mice that rely completely on shivering in the cold. We examine soleus muscle, which participates in shivering, and flexor digitorum brevis (FDB) muscle, a distal and superficial muscle that does not shiver. Soleus muscles of cold-acclimated UCP1-KO mice exhibited severe RyR1 PKA hyperphosphorylation and calstabin1 depletion, as well as markedly decreased SR Ca2+ release and force during contractions. In stark contrast, the RyR1 channel complexes were little affected, and Ca2+ and force were not decreased in FDB muscles of cold-acclimated UCP1-KO mice. These results indicate that activation of UCP1-mediated heat production in brown adipose tissue during cold exposure reduces the necessity for shivering and thus prevents the development of severe dysfunction in shivering muscles. Aydin, J., Shabalina, I. G., Place, N., Reiken, S., Zhang, S.-J., Bellinger, A. M., Nedergaard, J., Cannon, B., Marks, A. R., Bruton, J. D., Westerblad, H. Nonshivering thermogenesis protects against defective calcium handling in muscle.

Background Sudden infant death syndrome (SIDS) is the leading cause of postneonatal mortality in the United States. Mutations in the RyR2-encoded cardiac ryanodine receptor cause the highly lethal catecholaminergic polymorphic ventricular tachycardia (CPVT1) in the young. Objective The purpose of this study was to determine the spectrum and prevalence of RyR2 mutations in a large cohort of SIDS cases. Methods Using polymerase chain reaction, denaturing high performance liquid chromatography, and direct DNA sequencing, a targeted mutational analysis of RyR2 was performed on genomic DNA isolated from frozen necropsy tissue on 134 unrelated cases of SIDS (57 females, 77 males; 83 white, 50 black, 1 Hispanic; average age=2.7 months). RyR2 mutations were engineered by site-directed mutagenesis, heterologously expressed in HEK293 cells, and functionally characterized using single-channel recordings in planar lipid bilayers. Results Overall, two distinct and novel RyR2 mutations were identified in two cases of SIDS. A 6-month-old black female hosted an R2267H missense mutation, and a 4-week-old white female infant harbored a S4565R mutation. Both nonconservative amino acid substitutions were absent in 400 reference alleles, involved conserved residues, and were localized to key functionally significant domains. Under conditions that simulate stress [Protein Kinase A (PKA) phosphorylation] during diastole (low activating [Ca2+]), SIDS-associated RyR2 mutant channels displayed a significant gain-of-function phenotype consistent with the functional effect of previously characterized CPVT-associated RyR2 mutations. Conclusions Here we report a novel pathogenic mechanism for SIDS, whereby SIDS-linked RyR2 mutations alter the response of the channels to sympathetic nervous system stimulation such that during stress the channels become "leaky" and thus potentially trigger fatal cardiac arrhythmias.

The ryanodine receptor (RyR)/calcium-release channel on the sarcoplasmic reticulum mediates intracellular calcium release required for striated muscle contraction. RyR2, the predominant isoform in cardiac myocytes, comprises a macromolecular complex that includes calstabin2 (FKBP12.6). Calstabin2, an 11.8-kDa cis-trans peptidyl-prolyl isomerase (apparent molecular mass 12.6 kDa), stabilizes the closed state of the RyR2 channel, but the mechanism by which it achieves this regulation is not fully understood. Protein kinase A (PKA) phosphorylation of RyR2 decreases the affinity of calstabin2 for the RyR2 channel complex. In the present study we identified key aspartic acid residues on calstabin2 that are involved in binding to RyR2 and likely play a role in PKA phosphorylation-induced dissociation of calstabin2 from RyR2. We show that a mutant calstabin2 in which a key negatively charged residue (Asp-37) has been neutralized binds to a mutant RyR2 channel that mimics constitutively PKA-phosphorylated RyR2 (RyR2–S2808D). Furthermore, using wild-type and genetically altered murine models of heart failure induced by myocardial infarction, we show that manipulating the stoichiometry between calstabin2 and RyR2 can restore normal cardiac function in vivo .

Abnormalities in intracellular calcium release and reuptake are responsible for decreased contractility in heart failure (HF). We have previously shown that cardiac ryanodine receptors (RyRs) are protein kinase A-hyperphosphorylated and depleted of the regulatory subunit calstabin-2 in HF. Moreover, similar alterations in skeletal muscle RyR have been linked to increased fatigability in HF. To determine whether restoration of calstabin binding to RyR may ameliorate cardiac and skeletal muscle dysfunction in HF, we treated WT and calstabin-2 -/- mice subjected to myocardial infarction (MI) with JTV519. JTV519, a 1,4-benzothiazepine, is a member of a class of drugs known as calcium channel stabilizers, previously shown to increase calstabin binding to RyR. Echocardiography at 21 days after MI demonstrated a significant increase in ejection fraction in WT mice treated with JTV519 (45.8 ± 5.1%) compared with placebo (31.1 ± 3.1%; P < 0.05). Coimmunoprecipitation experiments revealed increased amounts of calstabin-2 bound to the RyR2 channel in JTV519-treated WT mice. However, JTV519 did not show any of these beneficial effects in calstabin-2 -/- mice with MI. Additionally, JTV519 improved skeletal muscle fatigue in WT and calstabin-2 -/- mice with HF by increasing the binding of calstabin-1 to RyR1. The observation that treatment with JTV519 improved cardiac function in WT but not calstabin-2 -/- mice indicates that calstabin-2 binding to RyR2 is required for the beneficial effects in failing hearts. We conclude that JTV519 may provide a specific way to treat the cardiac and skeletal muscle myopathy in HF by increasing calstabin binding to RyR.

The cardiac ryanodine receptor (RyR2)/calcium release channel on the sarcoplasmic reticulum is required for muscle excitation-contraction coupling. Using site-directed mutagenesis, we identified the specific Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) phosphorylation site on recombinant RyR2, distinct from the site for protein kinase A (PKA) that mediates the “fight-or-flight” stress response. CaMKII phosphorylation increased RyR2 Ca 2+ sensitivity and open probability. CaMKII was activated at increased heart rates, which may contribute to enhanced Ca 2+ -induced Ca 2+ release. Moreover, rate-dependent CaMKII phosphorylation of RyR2 was defective in heart failure. CaMKII-mediated phosphorylation of RyR2 may contribute to the enhanced contractility observed at higher heart rates. The full text of this article is available online at http://circres.ahajournals.org .

The inositol 1,4,5-trisphosphate receptor (IP3R) is a ubiquitously expressed intracellular calcium (Ca(2+)) release channel on the endoplasmic reticulum. IP3Rs play key roles in controlling Ca(2+) signals that activate numerous cellular functions including T cell activation, neurotransmitter release, oocyte fertilization and apoptosis. There are three forms of IP3R, all of which are ligand-gated channels activated by the second messenger inositol 1,4,5-trisphosphate. Channel function is modulated via cross-talk with other signaling pathways including those mediated by kinases and phosphatases. In particular IP3Rs are known to be regulated by cAMP-dependent protein kinase (PKA) phosphorylation. In the present study we show that PKA and the protein phosphatases PP1 and PP2A are components of the IP3R1 macromolecular signaling complex. PKA phosphorylation of IP3R1 increases channel activity in planar lipid bilayers. These studies indicate that regulation of IP3R1 function via PKA phosphorylation involves components of a macromolecular signaling complex.

Sympathetic nervous system (SNS) regulation of cardiac action potential duration (APD) is mediated by β adrenergic receptor (βAR) activation, which increases the slow outward potassium ion current ( I KS ). Mutations in two human I KS channel subunits, hKCNQ1 and hKCNE1, prolong APD and cause inherited cardiac arrhythmias known as LQTS (long QT syndrome). We show that βAR modulation of I KS requires targeting of adenosine 3′,5′-monophosphate (cAMP)–dependent protein kinase (PKA) and protein phosphatase 1 (PP1) to hKCNQ1 through the targeting protein yotiao. Yotiao binds to hKCNQ1 by a leucine zipper motif, which is disrupted by an LQTS mutation (hKCNQ1-G589D). Identification of the hKCNQ1 macromolecular complex provides a mechanism for SNS modulation of cardiac APD through I KS .

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.

Background Disruption of the sarcolemma-associated dystrophin-glycoprotein complex underlies multiple forms of muscular dystrophy, including Duchenne muscular dystrophy and sarcoglycanopathies. A hallmark of these disorders is muscle weakness. In a murine model of Duchenne muscular dystrophy, mdx mice, cysteine-nitrosylation of the calcium release channel/ryanodine receptor type 1 (RyR1) on the skeletal muscle sarcoplasmic reticulum causes depletion of the stabilizing subunit calstabin1 (FKBP12) from the RyR1 macromolecular complex. This results in a sarcoplasmic reticular calcium leak via defective RyR1 channels. This pathological intracellular calcium leak contributes to reduced calcium release and decreased muscle force production. It is unknown whether RyR1 dysfunction occurs also in other muscular dystrophies. Methods To test this we used a murine model of Limb-Girdle muscular dystrophy, deficient in β-sarcoglycan (Sgcb−/−). Results Skeletal muscle RyR1 from Sgcb−/− deficient mice were oxidized, nitrosylated, and depleted of the stabilizing subunit calstabin1, which was associated with increased open probability of the RyR1 channels. Sgcb−/− deficient mice exhibited decreased muscle specific force and calcium transients, and displayed reduced exercise capacity. Treating Sgcb−/− mice with the RyR stabilizing compound S107 improved muscle specific force, calcium transients, and exercise capacity. We have previously reported similar findings in mdx mice, a murine model of Duchenne muscular dystrophy. Conclusions Our data suggest that leaky RyR1 channels may underlie multiple forms of muscular dystrophy linked to mutations in genes encoding components of the dystrophin-glycoprotein complex. A common underlying abnormality in calcium handling indicates that pharmacological targeting of dysfunctional RyR1 could be a novel therapeutic approach to improve muscle function in Limb-Girdle and Duchenne muscular dystrophies.