Robert T. Dirksen

Reversal of RNA dominance by displacement of protein sequestered on triplet repeat RNA.

Resisting Repeats A set of diseases, including myotonic dystrophy, are caused by the expansion of a simple repeat in genomic DNA, which, when transcribed into RNA, can be toxic to other cellular processes. Ameliorating the effects of this toxic, repeat-laden RNA may also relieve the symptoms of the disease. Wheeler et al. (p. 336; see the Perspective by Cooper) developed an antisense morpholino oligonucleotide complementary to the expanded repeats found in the myotonic dystrophy protein kinase messenger RNA (mRNA). The morpholino bound the repeats in vitro and displaced the inappropriately bound and sequestered RNA splicing factor, Muscleblind-like 1. In an in vivo mouse model for myotonic dystrophy, local injection of the morpholino corrected a number of cellular defects in muscle, including the alternative mRNA splicing of several genes, among them the muscle-specific chloride channel, CIC1, leading to a marked reduction in the myotonia. An antisense oligonucleotide ameliorates the symptoms of myotonic dystrophy in transgenic mice. Genomic expansions of simple tandem repeats can give rise to toxic RNAs that contain expanded repeats. In myotonic dystrophy, the expression of expanded CUG repeats (CUGexp) causes abnormal regulation of alternative splicing and neuromuscular dysfunction. We used a transgenic mouse model to show that derangements of myotonic dystrophy are reversed by a morpholino antisense oligonucleotide, CAG25, that binds to CUGexp RNA and blocks its interaction with muscleblind-like 1 (MBNL1), a CUGexp-binding protein. CAG25 disperses nuclear foci of CUGexp RNA and reduces the overall burden of this toxic RNA. As MBNL1 is released from sequestration, the defect of alternative splicing regulation is corrected, thereby restoring ion channel function. These findings suggest an alternative use of antisense methods, to inhibit deleterious interactions of proteins with pathogenic RNAs.

Primary structure and functional expression of the ω-conotoxin-sensitive N-type calcium channel from rabbit brain

The complete amino acid sequence of a rabbit brain calcium channel (BIII) has been deduced by cloning and sequencing the cDNA. The open reading frame encodes 2339 amino acids, which corresponds to an M(r) of 261,167. A phylogenetic tree representing evolutionary relationships indicates that BIII is grouped together with the other rabbit brain calcium channels, BI and BII, into a subfamily that is distinct from the dihydropyridine-sensitive L-type subfamily. Transient expression in cultured skeletal muscle myotubes derived from muscular dysgenic mice demonstrates that the BIII channel mediates an omega-conotoxin-sensitive calcium current with kinetics and voltage dependence like those previously reported for whole-cell N-type current. Cell-attached patch recordings, with isotonic barium as the charge carrier, revealed distinct single channels with an average slope conductance of 14.3 pS.

RyR1 S-Nitrosylation Underlies Environmental Heat Stroke and Sudden Death in Y522S RyR1 Knockin Mice

Mice with a malignant hyperthermia mutation (Y522S) in the ryanodine receptor (RyR1) display muscle contractures, rhabdomyolysis, and death in response to elevated environmental temperatures. We demonstrate that this mutation in RyR1 causes Ca(2+) leak, which drives increased generation of reactive nitrogen species (RNS). Subsequent S-nitrosylation of the mutant RyR1 increases its temperature sensitivity for activation, producing muscle contractures upon exposure to elevated temperatures. The Y522S mutation in humans is associated with central core disease. Many mitochondria in the muscle of heterozygous Y522S mice are swollen and misshapen. The mutant muscle displays decreased force production and increased mitochondrial lipid peroxidation with aging. Chronic treatment with N-acetylcysteine protects against mitochondrial oxidative damage and the decline in force generation. We propose a feed-forward cyclic mechanism that increases the temperature sensitivity of RyR1 activation and underlies heat stroke and sudden death. The cycle eventually produces a myopathy with damaged mitochondria.

Identification of cysteines involved in S-nitrosylation, S-glutathionylation, and oxidation to disulfides in ryanodine receptor type 1.

The skeletal muscle Ca2+-release channel (ryanodine receptor type 1 (RyR1)) is a redox sensor, susceptible to reversible S-nitrosylation, S-glutathionylation, and disulfide oxidation. So far, Cys-3635 remains the only cysteine residue identified as functionally relevant to the redox sensing properties of the channel. We demonstrate that expression of the C3635A-RyR1 mutant in RyR1-null myotubes alters the sensitivity of the ryanodine receptor to activation by voltage, indicating that Cys-3635 is involved in voltage-gated excitation-contraction coupling. However, H2O2 treatment of C3635A-RyR1 channels or wild-type RyR1, following their expression in human embryonic kidney cells, enhances [3H]ryanodine binding to the same extent, suggesting that cysteines other than Cys-3635 are responsible for the oxidative enhancement of channel activity. Using a combination of Western blotting and sulfhydryl-directed fluorescent labeling, we found that two large regions of RyR1 (amino acids 1-2401 and 3120-4475), previously shown to be involved in disulfide bond formation, are also major sites of both S-nitrosylation and S-glutathionylation. Using selective isotopecoded affinity tag labeling of RyR1 and matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy, we identified, out of the 100 cysteines in each RyR1 subunit, 9 that are endogenously modified (Cys-36, Cys-315, Cys-811, Cys-906, Cys-1591, Cys-2326, Cys-2363, Cys-3193, and Cys-3635) and another 3 residues that were only modified with exogenous redox agents (Cys-253, Cys-1040, and Cys-1303). We also identified the types of redox modification each of these cysteines can undergo. In summary, we have identified a discrete subset of cysteines that are likely to be involved in the functional response of RyR1 to different redox modifications (S-nitrosylation, S-glutathionylation, and oxidation to disulfides).

The II-III Loop of the Skeletal Muscle Dihydropyridine Receptor Is Responsible for the Bi-directional Coupling with the Ryanodine Receptor

The dihydropyridine receptor (DHPR) in the skeletal muscle plasmalemma functions as both voltage-gated Ca2+ channel and voltage sensor for excitation-contraction (EC) coupling. As voltage sensor, the DHPR regulates intracellular Ca2+ release via the skeletal isoform of the ryanodine receptor (RyR-1). Interaction with RyR-1 also feeds back to increase the Ca2+ current mediated by the DHPR. To identify regions of the DHPR important for receiving this signal from RyR-1, we expressed in dysgenic myotubes a chimera (SkLC) having skeletal (Sk) DHPR sequence except for a cardiac (C) II-III loop (L). Tagging with green fluorescent protein (GFP) enabled identification of expressing myotubes. Dysgenic myotubes expressing GFP-SkLC or SkLC lacked EC coupling and had very small Ca2+currents. Introducing a short skeletal segment (α1Sresidues 720–765) into the cardiac II-III loop (replacing α1C residues 851–896) of GFP-SkLC restored both EC coupling and Ca2+ current densities like those of the wild type skeletal DHPR. This 46-amino acid stretch of skeletal sequence was recently shown to be capable of transferring strong, skeletal-type EC coupling to an otherwise cardiac DHPR (Nakai, J., Tanabe, T., Konno, T., Adams, B., and Beam, K.G. (1998) J. Biol. Chem.273, 24983–24986). Thus, this segment of the skeletal II-III loop contains a motif required for both skeletal-type EC coupling and RyR-1-mediated enhancement of Ca2+ current.

Epac and phospholipase Cepsilon regulate Ca2+ release in the heart by activation of protein kinase Cepsilon and calcium-calmodulin kinase II.

Recently, we identified a novel signaling pathway involving Epac, Rap, and phospholipase C (PLC)ϵ that plays a critical role in maximal β-adrenergic receptor (βAR) stimulation of Ca2+-induced Ca2+ release (CICR) in cardiac myocytes. Here we demonstrate that PLCϵ phosphatidylinositol 4,5-bisphosphate hydrolytic activity and PLCϵ-stimulated Rap1 GEF activity are both required for PLCϵ-mediated enhancement of sarcoplasmic reticulum Ca2+ release and that PLCϵ significantly enhances Rap activation in response to βAR stimulation in the heart. Downstream of PLCϵ hydrolytic activity, pharmacological inhibition of PKC significantly inhibited both βAR- and Epac-stimulated increases in CICR in PLCϵ+/+ myocytes but had no effect in PLCϵ–/– myocytes. βAR and Epac activation caused membrane translocation of PKCϵ in PLCϵ+/+ but not PLCϵ–/– myocytes and small interfering RNA-mediated PKCϵ knockdown significantly inhibited both βAR and Epac-mediated CICR enhancement. Further downstream, the Ca2+/calmodulin-dependent protein kinase II (CamKII) inhibitor, KN93, inhibited βAR- and Epac-mediated CICR in PLCϵ+/+ but not PLCϵ–/– myocytes. Epac activation increased CamKII Thr286 phosphorylation and enhanced phosphorylation at CamKII phosphorylation sites on the ryanodine receptor (RyR2) (Ser2815) and phospholamban (Thr17) in a PKC-dependent manner. Perforated patch clamp experiments revealed that basal and βAR-stimulated peak L-type current density are similar in PLCϵ+/+ and PLCϵ–/– myocytes suggesting that control of sarcoplasmic reticulum Ca2+ release, rather than Ca2+ influx through L-type Ca2+ channels, is the target of regulation of a novel signal transduction pathway involving sequential activation of Epac, PLCϵ, PKCϵ, and CamKII downstream of βAR activation.

Mice with the R176Q cardiac ryanodine receptor mutation exhibit catecholamine-induced ventricular tachycardia and cardiomyopathy

Mutations in the cardiac ryanodine receptor 2 (RyR2) have been associated with catecholaminergic polymorphic ventricular tachycardia and a form of arrhythmogenic right ventricular dysplasia. To study the relationship between RyR2 function and these phenotypes, we developed knockin mice with the human disease-associated RyR2 mutation R176Q. Histologic analysis of hearts from RyR2R176Q/+ mice revealed no evidence of fibrofatty infiltration or structural abnormalities characteristic of arrhythmogenic right ventricular dysplasia, but right ventricular end-diastolic volume was decreased in RyR2R176Q/+ mice compared with controls, indicating subtle functional impairment due to the presence of a single mutant allele. Ventricular tachycardia (VT) was observed after caffeine and epinephrine injection in RyR2R176Q/+, but not in WT, mice. Intracardiac electrophysiology studies with programmed stimulation also elicited VT in RyR2R176Q/+ mice. Isoproterenol administration during programmed stimulation increased both the number and duration of VT episodes in RyR2R176Q/+ mice, but not in controls. Isolated cardiomyocytes from RyR2R176Q/+ mice exhibited a higher incidence of spontaneous Ca2+ oscillations in the absence and presence of isoproterenol compared with controls. Our results suggest that the R176Q mutation in RyR2 predisposes the heart to catecholamine-induced oscillatory calcium-release events that trigger a calcium-dependent ventricular arrhythmia.

Epac-mediated activation of phospholipase C∈ plays a critical role in β-adrenergic receptor-dependent enhancement of Ca2+ mobilization in cardiac myocytes

Recently we demonstrated that PLCϵ plays an important role in β-adrenergic receptor (βAR) stimulation of Ca2+-induced Ca2+ release (CICR) in cardiac myocytes. Here we have reported for the first time that a pathway downstream of βAR involving the cAMP-dependent Rap GTP exchange factor, Epac, and PLCϵ regulates CICR in cardiac myocytes. To demonstrate a role for Epac in the stimulation of CICR, cardiac myocytes were treated with an Epac-selective cAMP analog, 8-4-(chlorophenylthio)-2′-O-methyladenosine-3′,5′-monophosphate (cpTOME). cpTOME treatment increased the amplitude of electrically evoked Ca2+ transients, implicating Epac for the first time in cardiac CICR. This response is abolished in PLCϵ-/- cardiac myocytes but rescued by transduction with PLCϵ, indicating that Epac is upstream of PLCϵ. Furthermore, transduction of PLCϵ+/+ cardiac myocytes with a Rap inhibitor, RapGAP1, significantly inhibited isoproterenol-dependent CICR. Using a combination of cpTOME and PKA-selective activators and inhibitors, we have shown that βAR-dependent increases in CICR consist of two independent components mediated by PKA and the novel Epac/PLCϵ pathway. We also show that Epac/PLCϵ-dependent effects on CICR are independent of sarcoplasmic reticulum loading and Ca2+ clearance mechanisms. These data define a novel endogenous PKA-independent βAR-signaling pathway through cAMP-dependent Epac activation, Rap, and PLCϵ that enhances intracellular Ca2+ release in cardiac myocytes.

Mitochondria are linked to calcium stores in striated muscle by developmentally regulated tethering structures.

Bi-directional calcium (Ca(2+)) signaling between mitochondria and intracellular stores (endoplasmic/sarcoplasmic reticulum) underlies important cellular functions, including oxidative ATP production. In striated muscle, this coupling is achieved by mitochondria being located adjacent to Ca(2+) stores (sarcoplasmic reticulum [SR]) and in proximity of release sites (Ca(2+) release units [CRUs]). However, limited information is available with regard to the mechanisms of mitochondrial-SR coupling. Using electron microscopy and electron tomography, we identified small bridges, or tethers, that link the outer mitochondrial membrane to the intracellular Ca(2+) stores of muscle. This association is sufficiently strong that treatment with hypotonic solution results in stretching of the SR membrane in correspondence of tethers. We also show that the association of mitochondria to the SR is 1) developmentally regulated, 2) involves a progressive shift from a longitudinal clustering at birth to a specific CRU-coupled transversal orientation in adult, and 3) results in a change in the mitochondrial polarization state, as shown by confocal imaging after JC1 staining. Our results suggest that tethers 1) establish and maintain SR-mitochondrial association during postnatal maturation and in adult muscle and 2) likely provide a structural framework for bi-directional signaling between the two organelles in striated muscle.

Sarcoplasmic reticulum: The dynamic calcium governor of muscle

The sarcoplasmic reticulum (SR) provides feedback control required to balance the processes of calcium storage, release, and reuptake in skeletal muscle. This balance is achieved through the concerted action of three major classes of SR calcium‐regulatory proteins: (1) luminal calcium‐binding proteins (calsequestrin, histidine‐rich calcium‐binding protein, junctate, and sarcalumenin) for calcium storage; (2) SR calcium release channels (type 1 ryanodine receptor or RyR1 and IP3 receptors) for calcium release; and (3) sarco(endo)plasmic reticulum Ca2+‐ATPase (SERCA) pumps for calcium reuptake. Proper calcium storage, release, and reuptake are essential for normal skeletal muscle function. We review SR structure and function during normal skeletal muscle activity, the proteins that orchestrate calcium storage, release, and reuptake, and how phenotypically distinct muscle diseases (e.g., malignant hyperthermia, central core disease, and Brody disease) can result from subtle alterations in the activity of several key components of the SR calcium‐regulatory machinery. Muscle Nerve, 2006