Calcium Channel Mutations in Cardiac Arrhythmia Syndromes.

Voltage gated calcium channels are essential for cardiac physiology by serving as sarcolemma- restricted gatekeepers for calcium in cardiac myocytes. Activation of the L-type voltagegated calcium channel provides the calcium entry required for excitation-contraction coupling and contributes to the plateau phase of the cardiac action potential. Given these critical physiological roles, subtle disturbances in L-type channel function can lead to fatal cardiac arrhythmias. Indeed, numerous human arrhythmia syndromes have been linked to mutations in the L-type channel leading to gain-of-function or loss-of-function mutations. In this review, we discuss the current state of knowledge regarding these mutations present in Timothy Syndrome, Long and Short QT Syndromes, Brugada Syndrome and Early Repolarization Syndrome. We discuss the pathological consequences of the mutations, the biophysical effects of the mutations on the channel as well as possible therapeutic considerations and challenges for future studies.

Mutations in SCN10A are responsible for a large fraction of cases of Brugada syndrome.

BACKGROUND: BrS is an inherited sudden cardiac death syndrome. Less than 35% of BrS probands have genetically identified pathogenic variants. Recent evidence has implicated SCN10A, a neuronal sodium channel gene encoding Nav1.8, in the electrical function of the heart. OBJECTIVES: The purpose of this study was to test the hypothesis that SCN10A variants contribute to the development of Brugada syndrome (BrS). METHODS: Clinical analysis and direct sequencing of BrS susceptibility genes were performed for 150 probands and family members as well as >200 healthy controls. Expression and coimmunoprecipitation studies were performed to functionally characterize the putative pathogenic mutations. RESULTS: We identified 17 SCN10A mutations in 25 probands (20 male and 5 female); 23 of the 25 probands (92.0%) displayed overlapping phenotypes. SCN10A mutations were found in 16.7% of BrS probands, approaching our yield for SCN5A mutations (20.1%). Patients with BrS who had SCN10A mutations were more symptomatic and displayed significantly longer PR and QRS intervals compared with SCN10A-negative BrS probands. The majority of mutations localized to the transmembrane-spanning regions. Heterologous coexpression of wild-type (WT) SCN10A with WT-SCN5A in HEK cells caused a near doubling of sodium channel current compared with WT-SCN5A alone. In contrast, coexpression of SCN10A mutants (R14L and R1268Q) with WT-SCN5A caused a 79.4% and 84.4% reduction in sodium channel current, respectively. The coimmunoprecipitation studies provided evidence for the coassociation of Nav1.8 and Nav1.5 in the plasma membrane. CONCLUSIONS: Our study identified SCN10A as a major susceptibility gene for BrS, thus greatly enhancing our ability to genotype and risk stratify probands and family members.

Stress-induced increase in skeletal muscle force requires protein kinase A phosphorylation of the ryanodine receptor.

Enhancement of contractile force (inotropy) occurs in skeletal muscle following neuroendocrine release of catecholamines and activation of muscle ß-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 ß-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.

Role of leaky neuronal ryanodine receptors in stress-induced cognitive dysfunction.

The type 2 ryanodine receptor/calcium release channel (RyR2), required for excitation-contraction coupling in the heart, is abundant in the brain. Chronic stress induces catecholamine biosynthesis and release, stimulating ß-adrenergic receptors and activating cAMP signaling pathways in neurons. In a murine chronic restraint stress model, neuronal RyR2 were phosphorylated by protein kinase A (PKA), oxidized, and nitrosylated, resulting in depletion of the stabilizing subunit calstabin2 (FKBP12.6) from the channel complex and intracellular calcium leak. Stress-induced cognitive dysfunction, including deficits in learning and memory, and reduced long-term potentiation (LTP) at the hippocampal CA3-CA1 connection were rescued by oral administration of S107, a compound developed in our laboratory that stabilizes RyR2-calstabin2 interaction, or by genetic ablation of the RyR2 PKA phosphorylation site at serine 2808. Thus, neuronal RyR2 remodeling contributes to stress-induced cognitive dysfunction. Leaky RyR2 could be a therapeutic target for treatment of stress-induced cognitive dysfunction.

Leaky ryanodine receptors in ß-sarcoglycan deficient mice: a potential common defect in muscular dystrophy.

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 ß-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.

Regulation of Ca²âº release through inositol 1,4,5-trisphosphate receptors by adenine nucleotides in parotid acinar cells.

Secretagogue-stimulated intracellular Ca(2+) signals are fundamentally important for initiating the secretion of the fluid and ion component of saliva from parotid acinar cells. The Ca(2+) signals have characteristic spatial and temporal characteristics, which are defined by the specific properties of Ca(2+) release mediated by inositol 1,4,5-trisphosphate receptors (InsP(3)R). In this study we have investigated the role of adenine nucleotides in modulating Ca(2+) release in mouse parotid acinar cells. In permeabilized cells, the Ca(2+) release rate induced by submaximal [InsP(3)] was increased by 5 mM ATP. Enhanced Ca(2+) release was not observed at saturating [InsP(3)]. The EC(50) for the augmented Ca(2+) release was ∼8 µM ATP. The effect was mimicked by nonhydrolysable ATP analogs. ADP and AMP also potentiated Ca(2+) release but were less potent than ATP. In acini isolated from InsP(3)R-2-null transgenic animals, the rate of Ca(2+) release was decreased under all conditions but now enhanced by ATP at all [InsP(3)]. In addition the EC(50) for ATP potentiation increased to ∼500 µM. These characteristics are consistent with the properties of the InsP(3)R-2 dominating the overall features of InsP(3)R-induced Ca(2+) release despite the expression of all isoforms. Finally, Ca(2+) signals were measured in intact parotid lobules by multiphoton microscopy. Consistent with the release data, carbachol-stimulated Ca(2+) signals were reduced in lobules exposed to experimental hypoxia compared with control lobules only at submaximal concentrations. Adenine nucleotide modulation of InsP(3)R in parotid acinar cells likely contributes to the properties of Ca(2+) signals in physiological and pathological conditions.

Mutation of the NADH oxidase gene (nox) reveals an overlap of the oxygen- and acid-mediated stress responses in Streptococcus mutans.

NADH oxidase (Nox) is a flavin-containing enzyme used by Streptococcus mutans to reduce dissolved oxygen encountered during growth in the oral cavity. In this study, we characterized the role of the NADH oxidase in the oxidative and acid stress responses of S. mutans. A nox-defective mutant strain of S. mutans and its parental strain, the genomic type strain UA159, were exposed to various oxygen concentrations at pH values of 5 and 7 to better understand the adaptive mechanisms used by the organism to withstand environmental pressures. With the loss of nox, the activities of oxygen stress response enzymes such as superoxide dismutase and glutathione oxidoreductase were elevated compared to those in controls, resulting in a greater adaptation to oxygen stress. In contrast, the loss of nox led to a decreased ability to grow in a low-pH environment despite an increased resistance to severe acid challenge. Analysis of the membrane fatty acid composition revealed that for both the nox mutant and UA159 parent strain, growth in an oxygen-rich environment resulted in high proportions of unsaturated membrane fatty acids, independent of external pH. The data indicate that S. mutans membrane fatty acid composition is responsive to oxidative stress, as well as changes in environmental pH, as previously reported (E. M. Fozo and R. G. Quivey, Jr., Appl. Environ. Microbiol. 70:929-936, 2004). The heightened ability of the nox strain to survive acidic and oxidative environmental stress suggests a multifaceted response system that is partially dependent on oxygen metabolites.

Ryanodine receptor oxidation causes intracellular calcium leak and muscle weakness in aging.

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.

Role of chronic ryanodine receptor phosphorylation in heart failure and ß-adrenergic receptor blockade in mice.

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, ß-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 ß-blocker therapy in HF.

Phosphorylation of the ryanodine receptor mediates the cardiac fight or flight response in mice.

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 ß-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.

InsP3R-associated cGMP kinase substrate determines inositol 1,4,5-trisphosphate receptor susceptibility to phosphoregulation by cyclic nucleotide-dependent kinases.

Ca(2+) release through inositol 1,4,5-trisphosphate receptors (InsP(3)R) can be modulated by numerous factors, including input from other signal transduction cascades. These events shape the spatio-temporal characteristics of the Ca(2+) signal and provide fidelity essential for the appropriate activation of effectors. In this study, we investigate the regulation of Ca(2+) release via InsP(3)R following activation of cyclic nucleotide-dependent kinases in the presence and absence of expression of a binding partner InsP(3)R-associated cGMP kinase substrate (IRAG). cGMP-dependent kinase (PKG) phosphorylation of only the S2+ InsP(3)R-1 subtype resulted in enhanced Ca(2+) release in the absence of IRAG expression. In contrast, IRAG bound to each InsP(3)R subtype, and phosphorylation of IRAG by PKG attenuated Ca(2+) release through all InsP(3)R subtypes. Surprisingly, simply the expression of IRAG attenuated phosphorylation and inhibited the enhanced Ca(2+) release through InsP(3)R-1 following cAMP-dependent protein kinase (PKA) activation. In contrast, IRAG expression did not influence the PKA-enhanced activity of the InsP(3)R-2. Phosphorylation of IRAG resulted in reduced Ca(2+) release through all InsP(3)R subtypes during concurrent activation of PKA and PKG, indicating that IRAG modulation is dominant under these conditions. These studies yield mechanistic insight into how cells with various complements of proteins integrate and prioritize signals from ubiquitous signaling pathways.

Linking structure to function: Recent lessons from inositol 1,4,5-trisphosphate receptor mutagenesis.

Great insight has been gained into the structure and function of the inositol 1,4,5 trisphosphate receptor (InsP(3)R) by studies employing mutagenesis of the cDNA encoding the receptor. Notably, early studies using this approach defined the key constituents required for InsP(3) binding in the N-terminus and the membrane spanning regions in the C-terminal domain responsible for channel formation, targeting and function. In this article we evaluate recent studies which have used a similar approach to investigate key residues underlying the in vivo modulation by select regulatory factors. In addition, we review studies defining the structural requirements in the channel domain which comprise the conduction pathway and are suggested to be involved in the gating of the channel.

Role of CaMKIIdelta phosphorylation of the cardiac ryanodine receptor in the force frequency relationship and heart failure.

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.

Ryanodine receptor studies using genetically engineered mice.

Ryanodine receptors (RyR) regulate intracellular Ca(2+) release in many cell types and have been implicated in a number of inherited human diseases. Over the past 15 years genetically engineered mouse models have been developed to elucidate the role that RyRs play in physiology and pathophysiology. To date these models have implicated RyRs in fundamental biological processes including excitation-contraction coupling and long term plasticity as well as diseases including malignant hyperthermia, cardiac arrhythmias, heart failure, and seizures. In this review we summarize the RyR mouse models and how they have enhanced our understanding of the RyR channels and their roles in cellular physiology and disease.

Ryanodine receptor channelopathies.

Ryanodine receptors (RyR) are intracellular Ca2+-permeable channels that provide the sarcoplasmic reticulum Ca2+ release required for skeletal and cardiac muscle contractions. RyR1 underlies skeletal muscle contraction, and RyR2 fulfills this role in cardiac muscle. Over the past 20 years, numerous mutations in both RyR isoforms have been identified and linked to skeletal and cardiac diseases. Malignant hyperthermia, central core disease, and catecholaminergic polymorphic ventricular tachycardia have been genetically linked to mutations in either RyR1 or RyR2. Thus, RyR channelopathies are both of interest because they cause significant human diseases and provide model systems that can be studied to elucidate important structure-function relationships of these ion channels.

Altered G-protein coupling in an mGluR6 point mutant associated with congenital stationary night blindness.

The highly specialized metabotropic glutamate receptor type 6 (mGluR6) is postsynaptically localized and expressed only in the dendrites of ON bipolar cells. Upon activation of mGluR6 by glutamate released from photoreceptors, a nonselective cation channel is inhibited, causing these cells to hyperpolarize. Mutations in this gene have been implicated in the development of congenital stationary night blindness type 1 (CSNB1). We investigated five known mGluR6 point mutants that lead to CSNB1 to determine the molecular mechanism of each phenotype. In agreement with other studies, four mutants demonstrated trafficking impairment. However, mGluR6 E775K (E781K in humans) suggested no trafficking or signaling deficiencies measured by our initial assays. Most importantly, our results indicate a switch in G-protein coupling, in which E775K loses G(o) coupling but retains coupling to G(i), which may explain the phenotype.

Protein kinase A increases type-2 inositol 1,4,5-trisphosphate receptor activity by phosphorylation of serine 937.

Protein kinase A (PKA) phosphorylation of inositol 1,4,5-trisphosphate receptors (InsP(3)Rs) represents a mechanism for shaping intracellular Ca(2+) signals following a concomitant elevation in cAMP. Activation of PKA results in enhanced Ca(2+) release in cells that express predominantly InsP(3)R2. PKA is known to phosphorylate InsP(3)R2, but the molecular determinants of this effect are not known. We have expressed mouse InsP(3)R2 in DT40-3KO cells that are devoid of endogenous InsP(3)R and examined the effects of PKA phosphorylation on this isoform in unambiguous isolation. Activation of PKA increased Ca(2+) signals and augmented the single channel open probability of InsP(3)R2. A PKA phosphorylation site unique to the InsP(3)R2 was identified at Ser(937). The enhancing effects of PKA activation on this isoform required the phosphorylation of Ser(937), since replacing this residue with alanine eliminated the positive effects of PKA activation. These results provide a mechanism responsible for the enhanced Ca(2+) signaling following PKA activation in cells that express predominantly InsP(3)R2.

ATP regulation of type-1 inositol 1,4,5-trisphosphate receptor activity does not require walker A-type ATP-binding motifs.

ATP is known to increase the activity of the type-1 inositol 1,4,5-trisphosphate receptor (InsP3R1). This effect is attributed to the binding of ATP to glycine rich Walker A-type motifs present in the regulatory domain of the receptor. Only two such motifs are present in neuronal S2+ splice variant of InsP3R1 and are designated the ATPA and ATPB sites. The ATPA site is unique to InsP3R1, and the ATPB site is conserved among all three InsP3R isoforms. Despite the fact that both the ATPA and ATPB sites are known to bind ATP, the relative contribution of these two sites to the enhancing effects of ATP on InsP3R1 function is not known. We report here a mutational analysis of the ATPA and ATPB sites and conclude neither of these sites is required for ATP modulation of InsP3R1. ATP augmented InsP3-induced Ca2+ release from permeabilized cells expressing wild type and ATP-binding site-deficient InsP3R1. Similarly, ATP increased the single channel open probability of the mutated InsP3R1 to the same extent as wild type. ATP likely exerts its effects on InsP3R1 channel function via a novel and as yet unidentified mechanism.