Takaaki Ikemoto

Generation and Characterization of Mutant Mice Lacking Ryanodine Receptor Type 3

The ryanodine receptor type 3 (RyR-3) functions as a Ca2+-induced Ca2+ release (CICR) channel and is distributed in a wide variety of cell types including skeletal muscle and smooth muscle cells, neurons, and certain non-excitable cells. However, the physiological roles of RyR-3 are totally unclear. To gain an insight into the function of RyR-3 in vivo, we have generated mice lacking RyR-3 by means of the gene targeting technique. The mutant mice thus obtained showed apparently normal growth and reproduction. Although Ca2+-induced Ca2+ release from intracellular Ca2+ stores of the mutant skeletal muscle differed in Ca2+ sensitivity from that of wild-type muscle, excitation-contraction coupling of the mutant muscle seemed to be normal. Moreover, we could not find any significant disturbance in the smooth muscle and lymphocytes from the mutant mice. On the other hand, the mutant mice showed increased locomotor activity, which was about 2-fold greater than that of the control mice. These results indicate that the loss of RyR-3 causes no gross abnormalities and suggest that the lack of RyR-3-mediated Ca2+ signaling results in abnormalities of certain neurons in the central nervous system.

Micro-dystrophin cDNA ameliorates dystrophic phenotypes when introduced into mdx mice as a transgene

The adeno-associated virus vector is a good tool for gene transfer into skeletal muscle, but the length of a gene that can be incorporated is limited. To develop a gene therapy for Duchenne muscular dystrophy, we generated a series of rod-truncated micro-dystrophin cDNAs: M3 (one rod repeat, 3.9 kb), AX11 (three rod repeats, 4.4 kb), and CS1 (four rod repeats, 4.9 kb). These micro-dystrophins, driven by a CAG promoter, were used to produce transgenic (Tg) mdx mice and all three micro-dystrophins were shown to localize at the sarcolemma together with the expression of dystrophin-associated proteins. Among them, CS1 greatly improved dystrophic phenotypes of mdx mice and contractile force of the diaphragm in particular was restored to the level of normal C57BL/10 mice. AX11 modestly ameliorated the dystrophic pathology, but, importantly, M3-Tg mdx mice still showed severe dystrophic phenotypes. These data suggest that the rod structure, and its length in particular, is crucial for the function of micro-dystrophin.

Novel bifunctional probe for radioisotope-free photoaffinity labeling: compact structure comprised of photospecific ligand ligation and detectable tag anchoring units

A novel method for radioisotope-free photoaffinity labeling was developed, in which a bifunctional ligand is connected to a target protein by activation of a photoreactive group, such as an aromatic azido or 3-trifluoromethyl-3H-diazirin-3-yl group, and identification of the ligated product is achieved by anchoring of a detectable tag through the Staudinger-Bertozzi reaction with an alkyl azido moiety that survives photolysis. The chemical ground of this method was confirmed using model compounds with the bifunctional group under photoirradiation in the presence of trapping agents for reactive intermediates. The utility of the method has been demonstrated by specific labeling of the catalytic portion of human HMG-CoA reductase.

α1-Syntrophin–deficient skeletal muscle exhibits hypertrophy and aberrant formation of neuromuscular junctions during regeneration

α1-Syntrophin is a member of the family of dystrophin-associated proteins; it has been shown to recruit neuronal nitric oxide synthase and the water channel aquaporin-4 to the sarcolemma by its PSD-95/SAP-90, Discs-large, ZO-1 homologous domain. To examine the role of α1-syntrophin in muscle regeneration, we injected cardiotoxin into the tibialis anterior muscles of α1-syntrophin–null (α1syn−/−) mice. After the treatment, α1syn−/− muscles displayed remarkable hypertrophy and extensive fiber splitting compared with wild-type regenerating muscles, although the untreated muscles of the mutant mice showed no gross histological change. In the hypertrophied muscles of the mutant mice, the level of insulin-like growth factor-1 transcripts was highly elevated. Interestingly, in an early stage of the regeneration process, α1syn−/− mice showed remarkably deranged neuromuscular junctions (NMJs), accompanied by impaired ability to exercise. The contractile forces were reduced in α1syn−/− regenerating muscles. Our results suggest that the lack of α1-syntrophin might be responsible in part for the muscle hypertrophy, abnormal synapse formation at NMJs, and reduced force generation during regeneration of dystrophin-deficient muscle, all of which are typically observed in the early stages of Duchenne muscular dystrophy patients.

FUNCTIONAL AND MORPHOLOGICAL FEATURES OF SKELETAL MUSCLE FROM MUTANT MICE LACKING BOTH TYPE 1 AND TYPE 3 RYANODINE RECEPTORS

1 We generated mice with targeted disruptions in the genes for both ryanodine receptor type 1 (RyR‐1) and type 3 (RyR‐3) to study the functional roles of RyR subtypes in skeletal muscle. 2 In permeabilized myocytes lacking both the RyRs, the Ca2+‐induced Ca2+ release (CICR) mechanism was completely lost, and caffeine failed to induce Ca2+ release. 3 Replacement of potassium methanesulphonate in an experimental intracellular solution with choline chloride resulted in Ca2+ release in the wild‐type muscle but not in the mutant muscle lacking RyR‐1. 4 The double‐mutant mice exhibited more severe muscular degeneration than RyR‐1‐deficient mice with formation of large vacuoles and swollen mitochondria while structural coupling between T‐tubules and the sarcoplasmic reticulum was retained. 5 These results demonstrate that CICR is mediated solely by RyR‐1 and RyR‐3 in skeletal muscle cells, and suggest that RyR‐1 is involved in Cl−‐induced Ca2+ release. The results also suggest the presence of molecular components other than RyRs responsible for the triad formation. RyR‐3 may have a role in the normal morphogenesis of skeletal muscle cells, although functionally it can be replaced by RyR‐1.

Dantrolene analogues revisited: general synthesis and specific functions capable of discriminating two kinds of Ca2+ release from sarcoplasmic reticulum of mouse skeletal muscle

The general synthesis of dantrolene analogues with various substituents on its phenyl ring has been developed via palladium-catalyzed cross-coupling reactions, the Stille or Suzuki reaction, as the key step. The effects of synthesized analogues have been evaluated by two kinds of Ca(2+) release modes from sarcoplasmic reticulum (SR) of mouse skeletal muscle fibers based on: (1) the measurement of twitch contraction caused by the physiological Ca(2+) release (PCR) of intact skeletal muscle and (2) the rate of Ca(2+)-induced Ca(2+) release (CICR) in saponin-treated skinned muscle fibers. Although dantrolene, a lead compound, inhibits both twitch contraction and CICR, some structurally modified analogues exhibit one or the other of these effects. The methoxy congener, GIF-0185, potently inhibits the twitch contraction without affecting the CICR, while GIF-0166 and GIF-0248, the ortho-nitro regioisomer and ortho, ortho-dinitro substituted analogues, respectively, doubly potentiate the CICR exclusively.

Properties of Ca2+ release induced by clofibric acid from the sarcoplasmic reticulum of mouse skeletal muscle fibres

To characterize the effect of clofibric acid (Clof) on the Ca2+ release mechanism in the sarcoplasmic reticulum (SR) of skeletal muscle, we analysed the properties of Clof‐induced Ca2+ release under various conditions using chemically skinned skeletal muscle fibres of the mouse. Clof (>0.5 mM) released Ca2+ from the SR under Ca2+‐free conditions buffered with 10 mM EGTA (pCa >8). Co‐application of ryanodine and Clof at pCa >8 but not ryanodine alone reduced the Ca2+ uptake capacity of the SR. Thus, Ca2+ release induced by Clof at pCa >8 must be a result of the activation of the ryanodine receptor (RyR). At pCa >8, (i) Clof‐induced Ca2+ release was inhibited by adenosine monophosphate (AMP), (ii) the inhibitory effect of Mg2+ on the Clof‐induced Ca2+ release was saturated at about 1 mM, and (iii) Clof‐induced Ca2+ release was not inhibited by procaine (10 mM). These results indicate that Clof may activate the RyR‐Ca2+ release channels in a manner different from Ca2+‐induced Ca2+ release (CICR). In addition to this unique mode of opening, Clof also enhanced the CICR mode of opening of RyR‐Ca2+ release channels. Apart from CICR, a high concentration of Ca2+ might also enhance the unique mode of opening by Clof. These results suggest that some features of Ca2+ release activated by Clof are similar to those of physiological Ca2+ release (PCR) in living muscle cells and raise the possibility that Clof may be useful in elucidating the mechanism of PCR in skeletal muscle.

Effect of calmodulin on Ca2+-induced Ca2+ release of skeletal muscle from mutant mice expressing either ryanodine receptor type 1 or type 3.

Abstract We analyzed the effects of calmodulin (CaM) on Ca2+-induced Ca2+ release (CICR) in mouse skeletal muscle cells expressing only ryanodine receptor type 1 (RyR-1) or type 3 (RyR-3) following targeted disruption of one of the RyR genes. Under Mg2+-free conditions, CaM potentiated CICR via RyR-3 at low Ca2+ concentrations (pCa≥6) but inhibited CICR at high Ca2+ concentrations (pCa≤5). On the other hand, CaM potentiated CICR via RyR-1 between pCa 7 and pCa 5. Greater concentrations of CaM were required for potentiation of CICR at pCa 6 than for the inhibition at pCa 5 in the RyR-3-expressing cells. Similarly, higher concentrations of CaM were required for the potentiation of CICR via RyR-1 at pCa 6 than potentiation at pCa 5. In the presence of Mg2+ and β,γ-methyleneadenosine 5′-trisphosphate (AMPOPCP), the same differential effects of CaM on the CICR via the different subtypes of RyR were observed. These data suggest that multiple CaM-binding sites are involved in the differential effects on RyR-1 and RyR-3. These effects of CaM are important for the evaluation of the physiological roles of RyRs.

Effects of dantrolene and its derivatives on Ca2+ release from the sarcoplasmic reticulum of mouse skeletal muscle fibres

We analysed the effect of dantrolene (Dan) and five newly synthesized derivatives (GIFs) on Ca2+ release from the sarcoplasmic reticulum (SR) of mouse skeletal muscle. In intact muscles, GIF‐0185 reduced the size of twitch contraction induced by electrical stimulation to the same extent as Dan. GIF‐0082, an azido‐functionalized Dan derivative, also inhibited twitch contraction, although the extent of inhibition was less than that of Dan and of GIF‐0185. In skinned fibres, Dan inhibited Ca2+‐induced Ca2+ release (CICR) under Mg2+‐free conditions at room temperature. In contrast, GIF‐0082 and GIF‐0185 showed no inhibitory effect on CICR under the same conditions. Dan‐induced inhibition of CICR was not affected by the presence of GIF‐0082, whereas it was diminished in the presence of GIF‐0185. GIF‐0082 and GIF‐0185 significantly inhibited clofibric acid (Clof)‐induced Ca2+ release, as did Dan. Several Dan derivatives other than GIF‐0082 and GIF‐0185 showed an inhibitory effect on twitch tension but not on the CICR mechanism. All of these derivatives inhibited Clof‐induced Ca2+ release. The magnitudes of inhibition of Clof‐induced Ca2+ release by all Dan derivatives were well correlated with those of twitch inhibition. This supports the notion that the mode of Clof‐induced opening of the RyR‐Ca2+ release channel may be similar to that of physiological Ca2+ release (PCR). These results indicate that the difference in opening modes of the RyR‐Ca2+ release channel is recognized by certain Dan derivatives.

Morphological abnormalities of adrenal gland and hypertrophy of liver in mutant mice lacking ryanodine receptors

Abstract.Ryanodine receptors (RyRs), which form Ca2+ channels in the membrane of the endoplasmic reticulum, consist of three subtypes (RyR1, RyR2, and RyR3). The RyRs release Ca2+ from the endoplasmic reticulum into the cytoplasm and thus play an important role, especially in the contraction of skeletal and cardiac muscle cells. The genes of these RyRs are also expressed in many non-muscle tissues, but the role played by RyRs in non-muscle cells is not fully understood. In the present study, we examined the morphological changes in such cells caused by a deficiency of RyRs genes using three mutant mice lacking RyR1, RyR3, or both RyR1 and RyR3. The results showed morphological abnormalities in the adrenal cortical cells in all three mutant mice. In addition, an excessive accumulation of glycogen granules in hepatic cells, and a hypertrophy of the liver were both present in those mutant mice lacking both RyR1 and RyR3. We discuss the relationship between the morphological abnormalities of the adrenal cortex and liver induced by a deficiency of RyRs, and the possible causes of these abnormalities.