RNA binding motif 20 (RBM20) regulates pre-mRNA splicing of over thirty genes, among which titin is a major target. With RBM20 expression, titin expresses a larger isoform at fetal stage to a smaller isoform at adult resulting from alternative splicing, while, without RBM20, titin expresses exclusively a larger isoform throughout all ages. In addition to splicing regulation, it is unknown whether RBM20 also regulates gene expression. In this study, we employed Rbm20 knockout rats to investigate gene expression profile using Affymetrix expression array. We compared wild type to Rbm20 knockout at day1, 20 and 49. Bioinformatics analysis showed RBM20 regulates fewer genes expression at younger age and more at older age and commonly expressed genes have the same trends. GSEA indicated up-regulated genes are associated with heart failure. We examined titin binding partners. All titin direct binding partners are up-regulated and their increased expression is associated with dilated cardiomyopathy. Particularly, we found that genes involving calcium handling and muscle contraction are changed by RBM20. Intracellular calcium level measurement with individual cardiomyocytes further confirmed that changes of these proteins impact calcium handling. Selected genes from titin binding partners and calcium handling were validated with QPCR and western blotting. These data demonstrate that RBM20 regulates gene splicing as well as gene expression. Altered gene expression by RBM20 influences protein-protein interaction, calcium releasing and thus muscle contraction. Our results first reported gene expression impacted by RBM20 with heart maturation, and provided new insights into the role of RBM20 in the progression of heart failure.
Pre-mRNA splicing is an important biological process that contributes to protein diversity in eukaryotic organisms . This process is determined by RNA-binding splicing factors to produce protein isoforms in a tissue-specific and developmental-regulated manner . Tissue-specific alternative splicing is usually regulated by tissue-specific splicing factors or a combination with ubiquitously expressed splicing factors to influence spliceosome assembly at splice sites . Therefore, identification of tissue-specific splicing factors and their targets is essential to decipher the mechanism of pre-mRNA splicing. In muscle tissues, only a few splicing factors have been reported , and RNA binding motif 20 (RBM20) is one of the muscle tissue specific splicing factors that has been identified recently . Protein RBM20 contains two highly conserved functional domains, an RNA-recognition domain 1 (RRM1) and a serine (S)/arginine (R) rich domain (SR-domain) . These structural features defined it as a serine/arginine (SR) protein that is essential for assembly in the spliceosome of pre-mRNA splicing [6, 7]. RBM20 is highly expressed in striated muscles, with the highest expression levels in cardiac muscle . Rbm20 deficiency in animal models leads to dilated cardiomyopathy, a major cause of heart failure and a significant source of mortality and morbidity worldwide [4, 8], and mutations in RBM20 have been identified in human patients with familial dilated cardiomyopathy [4, 5, 9, 10, 11, 12, 13]. Given the role of RBM20 in the regulation of gene splicing, efforts toward the identification of its RNA targets have been made [4, 14] with a goal that these findings would shed light on human heart failure progression and provide strategies for molecular therapy. With these efforts, more than thirty RNA targets of RBM20 have been identified with a major target being titin [4, 14].
Titin is a giant multifunctional protein in the sarcomere that plays a critical role in elastic recoil of cardiac myocytes and contributes to passive stiffness of the myocardium [15-16, 17-18]. It has been increasingly recognized as a major disease-causing gene [16, 19]. Titin runs through distinct sarcomere regions: the Z-band, the I-band, the A-band and the M-band regions [20, 21]. Over twenty proteins have been found directly bound to these regions of titin, and over fifty proteins are indirectly bound to titin, linking it to multiple potential mechanic signaling pathways [22, 23]. Cardiac titin is encoded by a single gene, and it has two major classes of isoforms resulting from alternative splicing. These isoforms have been termed "N2B" and "N2BA" . The ratio changes of these two isoforms are a major determinant for diastolic passive stiffness during left ventricular filling [15-18, 25]. Aberrant ratio changes of titin isoforms have been found in both systolic and diastolic heart failure [1, 26-32]. Therefore, the role of RBM20 in the regulation of titin alternative splicing may be critical for correction of abnormal titin isoform changes, and thereby, the treatment of diastolic dysfunction. In addition to its major role of the regulation in titin splicing, it is unknown whether RBM20 also regulates gene expression profiles in the heart. In this study, we compared the gene expression profiles of wild type and the Rbm20 knockout rats, and demonstrated that calcium handling channel proteins are affected and regulated by RBM20.
Sprague-Dawley strain rats were crossed with Fisher 344 inbreeds. Both strains were obtained from Harlan Sprague Dawley, Indianapolis, IN. Animals were maintained on standard rodent chow using protocols approved by the University of Wisconsin-Madison and University of Wyoming Animal Use and Care Committees. Hearts were removed immediately after euthanasia and the left ventricle, right ventricle, and atria were separated. Samples were obtained from animals ranging from 16 days fetuses (E16) to 1 year after birth. Tissues were snap-frozen in liquid nitrogen and stored in -80°C freezer prior to gel sample and RNA preparation. All samples for protein gel electrophoresis were prepared as described previously .
Titin isoforms were resolved using a previously published method using a vertical sodium dodecyl sulfate (SDS)-1% agarose gel electrophoresis (VAGE) system . The frozen tissue samples from E16, E18 (18 days fetuses), day (d) 1, 10, 20, 30, 60, 180, and 1 year after birth were dissolved in urea-thiourea-SDS-dithiothreitol sample buffer using a small Dounce homogenizer or a MiniBeadBeater (Biospec Products, Bartlesville, OK) and were heated for 10min at ~65℃. Silver stained agarose gels were dried between sheets of mylar and dried gels were scanned. Human cardiac titin N2B (or the identically sized rat N2B) were used as standards to estimate molecular weights of the rat N2BA titin isoform. Full details have been discussed in a previous published report .
Total RNA was extracted in 1ml TRIzol (Invitrogen, Frederick, MD), following manufacturer instructions. RNA was precipitated from the TRIzol reagent with isopropanol and resuspended at 0.5-1.0mg/ml using RNA secure solution (Ambion, Austin, TX). RNA samples were treated with DNaseI (DNA-free; Ambion, Austin, TX). After extraction, all samples were stored at -80 degree. RNA concentration was measured with a NanoDrop ND 1000 spectrometer (NanoDrop Technologies, Wilmington, DE), and RNA integrity was assessed on an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). RNA was further purified by the RNeasy mini kit (Qiagen, Valencia, CA). Double stranded cDNA was synthesized from total RNA (SuperScript II system; Invitrogen). An in vitro transcription reaction was then performed to obtain biotin-labeled cRNA from the double-stranded cDNA (Enzo BioArray High Yield RNA Transcript Labeling kit; Enzo Diagnostics, Farmingdale, NY). The cRNA was fragmented before hybridization, and then mixed in a hybridization mixture containing probe array controls, BSA, and herring sperm DNA . A cleanup procedure was performed on the hybridization cocktail using an RNeasy spin column (Qiagen), after which it was applied to the Affymetrix Rat 230 2.0 probe array. A total of eighteen hybridization experiments were performed in which each stage (d1, d20 and d49) was represented by three wild type (Wt) and three RBM20 homozygous knockout (Hm) individual ventricular RNA extracts (Fig. 2A). Hybridization was allowed to continue for 16 h at 45°C in a GeneChip 640 hybridization oven, after which the arrays were washed and stained with phycoerythrin-conjugated streptavidin (Molecular Probes, Eugene, OR). Images were scanned using a GeneArray scanner (Agilent Technologies, Palo Alto, CA) and GeneChip cel files were subsequently processed by the log scale robust multi-array analysis (RMA). The RMA estimates were based upon a robust average of log2 (B (PM)), where B (PM) is background corrected perfect match intensities. Compared with the GCOS method the RMA method has better precision, provided more consistent estimates of fold change and provided higher specificity and sensitivity when using fold change analysis to detect differential expression .
Rats were injected with heparin (1000U/kg) 20mins before anesthetized with 60mg/kg ketamine and 8mg/kg xylazine (i.p.). Myocytes were isolated as described previously . In brief, under anesthesia, the heart was dissected and cannulated via the aorta. The heart was perfused at 37°C for 4 min with perfusion buffer (in mM: 113 NaCl, 4.7 KCl, 0.6 Na2HPO4, 1.2 MgSO4, 12 NaHCO3, 10 KHCO3, 10 HEPES, and 30 taurine, pH 7.4) equilibrated with 5%CO2-95% O2, followed by perfusion with digestion buffer (perfusion buffer plus 0.45 mg/ml collagenase II [240 U/mg; Worthington Biochemical Corporation], 0.13 mg/ml trypsin, and 25 µM CaCl2) for 8-10 min. When the heart was flaccid, digestion was halted and the heart was placed in myocyte stopping buffer 1 (perfusion buffer plus 0.04 ml bovine calf serum [BCS]/ml buffer and 5 µM CaCl2). The left ventricle (LV) was cut into small pieces, and the rest of the heart was discarded. The small pieces were then triturated several times with a transfer pipette and then filtered through a 300µm nylon mesh filter. After this, the cells were gravity pelleted and the supernatant was discarded. Next, 10 ml of myocyte stopping buffer 2 was added (perfusion buffer plus 0.05 ml BCS/ml buffer and 12.5 µM CaCl2), and Ca2+ was reintroduced to a final concentration of 1.8 mM. Then, myocytes were loaded with Fura-2 AM (0.5 μM) for 10 min, and fluorescence measurements were recorded with a dual-excitation fluorescence photomultiplier system (IonOptix). Myocytes were placed on an Olympus IX-70 inverted microscope stage and imaged through a Fluor 40× oil objective. Myocytes were exposed to light emitted by a 75-W lamp and passed through either a 360- or a 380-nm filter while being stimulated to contract at 0.5 Hz. Fluorescence emissions were detected between 480 and 520 nm by a photomultiplier tube after first illuminating the myocytes at 360 nm for 0.5 sec and then at 380 nm for the duration of the recording protocol (333Hz sampling rate). The excitation scan at 360 nm was repeated at the end of the protocol and qualitative changes in intracellular Ca2+ concentration were inferred from the ratio of Fura-2 fluorescence intensity (FFI) at the two wavelengths (360/380). Fluorescence decay time was measured as an indication of the intracellular Ca2+ clearing rate. Single exponential curve fit programs were used to derive the intracellular Ca2+ decay constant .
Total RNA was extracted using TRIzol, as described above. For reverse transcription, 60 ng of RNA were mixed with 5 uM random hexamers, 1 mM each dNTP, 7.5 mM MgCl2, 40 U RNasin (Promega, Madison, WI), 1X PCR buffer II (Applied Biosystems, Foster City, CA) and 250 U of SuperScript II reverse transcriptase (Invitrogen). The reaction mixture was incubated at 25°C for 10 min, 48°C for 45 min, and 95°C for 5 min, then cooled down to 4°C. Two volumes of ethanol were added to the cDNA and evenly mixed, and then stored at -20°C for 30 minutes, centrifuging for 15 minutes at 13,000 rpm, washed with 75% ethanol and dried. The cDNA was resuspended in distilled water and used as a template. For SYBR Green quantitative real-time PCR, all primers were designed from the selected genes (Supplement table S1). All primer pairs produced a single PCR product as determined by the dissociation curve and gel analysis. Real-time PCR was performed in a 20ul reaction, 96-well format and 1 X SYBR PCR Master Mix (Biorad). The reaction was incubated in a CFX96 real-time PCR system (Biorad) for 40 cycles consisting of denaturation at 95°C for 15 s and annealing/extension at 58-60°C for 1 min. Three samples were measured in each experimental group in quadruplicate, with a minimum of two independent experiments. The relative amount of target mRNA normalized to GAPDH was calculated according to the method described previously .
Total protein was separated by SDS-PAGE gel, and transferred onto a PVDF membrane. The membrane was probed with antibodies against FHL (Santa Cruz), CRP2 (Santa Cruz), IGFBP8/CTGF (Abcam), KCNQ1 (Santa Cruz), TGF β2 (Cell Signaling), RyR2 (Abcam), SERCA2A (Santa Cruz), TRDN (Santa Cruz), CASQ1 (Santa Cruz), PLB (Abcam), Rabbit anti-mouse IgG-conjugated with horseradish peroxidase (Fisher Scientific) was served as the secondary antibody. GAPDH (Santa Cruz) was served as the protein loading control. The detailed procedure was described previously .
GraphPad prism software was used for statistical analysis. Results were expressed as means ± SEM. Statistical significance between groups was determined using one way ANOVA with a Tukey post test, or a 2-tailed t-test for comparison of two groups. The significance level was P<0.05, P<0.01 and P<0.001.
A major RBM20 target, titin is a striated muscle-specific giant elastic protein and largely responsible for the generation of the diastolic force in the cardiac myocyte [15-18]. Titin has two classes of isoforms with one smaller isoform N2B that is stiffer, and the larger isoforms N2BAs that are more compliant [24-25]. Cardiac titin undergoes a series of isoform transitions during maturation in wild type rat (Wt) heart as observed by SDS agarose electrophoresis (embryonic day (E) 16, E18, day (d) 1, d10, d20, d30, d60, d180 and year (yr) 1) (Fig. 1A). Larger fetal titin N2BA isoform is predominantly expressed before E18, and decreases in sizes and amounts with maturation, while smaller N2B isoform starts to be present after E18 and dominant after d10 (Fig. 1A and 1C). Percentage of N2B to total titin (N2B+N2BA) changes from about 2% at E18 to approximately 85% at adult (Fig 1C). However, in Rbm20 knockout rats (Hm), titin is expressed as a constant size that is similar to the larger fetal titin N2BA isoform through all ages when compared to Wt rats (Fig. 1B and 1C). This constantly expressed larger titin isoform in Rbm20 Hm rats leads to reduced ventricular wall stiffness and has been associated with heart failure progression as described previously . Immunocytochemistry staining of cardiomyocytes isolated from adult rat hearts with RBM20 antibody indicated that RBM20 is located in splicing speckles in the nucleus with two spots in Wt cardiomyocytes and completely depleted in Hm cardiomyocytes (Fig 1D). Splicing speckles are nuclear bodies that are important for splicing factor storage and modification and contain components of the splicing machinery and polyA RNA [39-40].
Rbm20 deficient rat model. (A and B), Agarose gel showing titin protein isoform changes with development in wild type (Wt) and homozygous Rbm20 knockout (Hm); (C), Percentage of N2B with development in Wt and Hm; (D), Rbm20 location and expression in Wt and Hm rat adult cardiomyocytes. The arrows indicate the Rbm20 localization in the nucleus. Wt, wild type; Hm, homozygous Rbm20 knockout; E, embryonic day; D, day; yr, year.
Gene expression profile analysis with gene-chip data. (A), Biological replicates of heart tissues of Wt and Hm with development; (B), Venn Diagram analysis of differentially expressed genes at three ages; (C), Percentage of DEG with up- and down-regulated genes at three ages; The Heatmap indicated the up- and down-regulated genes at three ages between Wt and Hm. Wt, wild type; Hm, homozygous Rbm20 knockout; D, day; DEG, differentially expressed genes; Up, up-regulation; Down, down-regulation.
Our previous study has shown that Rbm20 deficiency in the rat model leads to heart failure with reduced ejection fraction, enlarged ventricular chamber and thinner ventricular walls . Additionally, Ours and other studies have identified over thirty RBM20 target genes that undergo alternative splicing [4, 14]. However, it is unknown whether Rbm20 deficiency leads to gene expression changes in the heart. Therefore, this study employs the Rbm20 knockout rat model to investigate gene expression profile using Affymetrix gene expression microarray and determine which genes or signaling pathways are associated with heart failure progression in Rbm20 knockout rats.
In order to examine the effect of RBM20 on gene differential expression in heart muscle at distinct developmental stages, we selected d1, d20 and d49 as representative maturation time courses according to titin isoform expression pattern as indicated in Fig. 1. The experimental design is shown in Fig 2A. Gene expression array data were analyzed based on student t test between Wt and Hm with p value cutoff less than 0.05 and fold change more than 1.5. Out of a total 31099 probes on the Affymetrix Rat 230 2.0 Chip, 418 probes representing 381 genes including 21 unannotated genes were differentially changed when comparing Hm to Wt rats (Supplement table S2). Among 381 genes, 77 of them are differentially expressed at d1 (Supplement table S3), 121 at d20 (Supplement table S4), and 227 at d49 (Supplement table S5). Venn diagram analysis of differentially expressed genes at three ages demonstrated that four genes were commonly expressed at d1 and 20, seven at d1 and 49, forty-nine at d20 and 49, and ten at all three ages (Fig. 2B). These data hint that most of differentially expressed genes regulated by RBM20 are development dependent. RBM20 regulates the expression of fewer genes at early developmental stages when compared to late stages, which matches the pattern of titin size changes from the larger ones to smaller during maturation. Interestingly, we found that almost 99% of differentially expressed genes are up-regulated at early maturation stage, and percentage of up-regulated genes decreases at d20 to approximately 68% and at d49 to 57% (Fig. 2C). These data suggest that genes regulated by RBM20 are fewer at d1 with mostly elevated expression by comparing to late maturation stages d20 and d49 with more down-regulated genes. However, gene expression pattern regulated by RBM20 at d20 and d49 are similar, which is consistent with titin splicing pattern with maturation (Fig. 1A).
Next we did further data mining for the relevant function of these differentially expressed genes. Gene Set Enrichment Analysis (GSEA) is a computational method that determines whether a priori defined set of genes shows statistically significant, concordant differences between two biological states . In order to interpret gene expression data by analyzing gene sets, GSEA analysis indicated that gene sets associated with failing heart were up-regulated at all three ages (Fig. 3A, 3B and 3C). Gene Ontology (GO) analysis indicated that most of differentially expressed genes were associated with protein binding and cell signal transduction (Fig. 3D). Gene cluster analysis suggested that the expression pattern of d20 and d49 between Wt and Hm was similar, but it was different from d1. GO and gene cluster analysis further confirmed that gene expression pattern regulated by RBM20 was consistent with titin splicing pattern changes in the heart of Wt rats which was also modulated by RBM20 (Fig. 3E, 1A and 1B).
Titin binds over 70 proteins directly and indirectly . It is unknown whether differentially expressed genes regulated by RBM20 contain titin binding partners. We searched the list of differentially expressed genes with known titin partners, and found that among these genes, proteins encoded by five of them directly bound to titin and twenty-two of them were indirectly associated with titin (Fig. 4). Five direct binding proteins were MyBP-C, Fhl1, Carp, Darp, and β-myosin. MyBPC has three isoforms: slow skeletal, fast skeletal and cardiac isoform that are encoded by three different genes [42-45]. Gene MyBPC2 encodes a fast skeletal isoform (fMyBP-C) that is expressed primarily in fast skeletal muscles . However, it can also be expressed in cardiac muscle, and its increased expression in the heart is associated with severe dilated cardiomyopathy . Mammalian cardiac muscle contains two myosin isoforms: α-myosin and β-myosin encoded by two distinct genes . In adult human cardiac muscle, the major isoform is β-myosin . Elevated β-myosin level has been found in human failing heart myocardium, cardiomyopathy, aging and pressure overload [49-51]. Four-and-a-half Lim domain protein-1 (Fhl1) is a member of Fhl family structurally characterized by four complete LIM domain and a N-terminal half LIM domain , which has been found associated with titin N2B domain  and the mitogen-activated protein kinase signaling cascade (Raf1/Mek1/2/Erk2) . Fhl1 plays an important role in sensing biomechanical hypertrophic stress responses in the sarcomere via titin N2B, which in turn regulates MAPK/hypertrophic signaling and muscle compliance [54-55]. Carp and Darp are two members of the three homologous muscle-ankyrin-repeat proteins (MARPs) that interact with titin I-band N2A region [56-57]. They are associated with muscle stress signaling and up-regulated when muscles undergo mechanical or metabolic challenge . Carp and Darp exist both in the nucleus and in the sarcomeric I-band, and show increased expression levels in end-stage failing human dilated cardiomyopathy hearts . All of these five direct titin binding partners were up-regulated in Rbm20 knockout rats. According to previous studies described above, up-regulation of these five genes in human heart or animal models leads to heart failure with preserved ejection fraction [46,49, 50, 55, 56], which was consistent with the phenotype of Rbm20 knockout rats and human patients with Rbm20 loss-of-function mutations .
In addition to titin direct binding partners, we found more titin indirect binding partners that are regulated by RBM20. These indirect binding partners play diverse roles in different physiological function. For example, Kcnq1, Kcnd2, Kcnd3 and Kcnk2 are potassium voltage-gated channels required for cardiac action potential, cellular excitability and heart function [58-61]. The cardiac gene expression and plasma level of ANP and BNP are in response to changes of diuresis, natriuresis and systemic blood pressure. Increased levels of ANP and BNP are associated with chronic heart failure, and can be predictors for left ventricular ejection fraction, mortality and morbidity in human patients [62-63]. Another example is Myl4 that encodes a protein called atrial light chain 1 (ALC-1). Increased expression of ALC-1 was found in patients with hypertrophic obstructive cardiomyopathy, and in ischemic and idiopathic cardiomyopathy with larger individual differences [51, 64-65]. RBM20-regulated genes involving diverse functions indicate that Rbm20 deficiency leads to heart failure through cooperation of multiple biological processes instead of single gene effect such as titin, though they may play minor roles when compared to titin.
Gene functional annotation and heatmap of pairwise correlations between Wt and Hm. (A, B and C), Gene Set Enrichment analysis indicated that gene sets associated with heart failure are up-regulated at all three ages; (D), A cluster of differentially expressed gene between Wt and Hm at three ages; (E), Molecular function of differentially expressed genes with Gene Ontology (GO) analysis. D, day; Wt, wild type; Hm, homozygous Rbm20 knockout; GO, Gene Ontology.
We next validated gene expression resulting from DNA microarray by QPCR and western blotting. We selected all five genes (Myosin, Carp, Darp, Mybpc2 and Fhl1) from direct titin binding partners and five genes (Myot, Anp, Myl4, Crp2 and Kcnq1) from indirect titin binding partners. We also selected six genes (Tgfβ2, Triadin/Trdn, Akip1, Eif4ebp, Igfbp8/Ctgf and Casq1) that haven't been identified associated with titin binding for validation at d1, d20 and d49. Comparisons were made between gene expression levels from DNA microarray and QPCR at individual ages. For titin direct binding partners, we found that all five genes were up-regulated and validated by QPCR at three ages which is consistent with DNA microarray data (Fig. 5A, 5B and 5C). For titin indirect binding partners, DNA microarray data indicated that all five genes were not increased by comparing to Wt at d1, while one gene Anp was validated to increase by QPCR at d1 (Fig. 5D). All five genes were significantly increased at d20 and d49 from both DNA microarray and QPCR (Fig. 5E and 5F). For other genes which are not related to titin binding, the expression level of genes Akip1 and Eif4ebp was similar to Wt at d1 from both DNA microarray and QPCR data. Expression of Igfbp8 was slightly lower by comparing to Wt at d1 from DNA microarray data, but it had no significant difference validated from QPCR. Gene Casq1 was slightly increased, but no significant difference at d1 from DNA microarray data, while QPCR results indicated significant increase. The expression level of Tgfβ2 and Trdn validated by QPCR was consistent with DNA microarray data. Interestingly, Genes Trdn and Eif4ebp were significantly decreased and Tgfβ2, Akip1, Igfbp8 and Casq1 were significantly increased by comparing to Wt at d20 and d49 from DNA microarray, which were confirmed by QPCR (Fig. 5G, 5H and 5I). We then selected total six proteins (Fhl1, Darp, Crp2, Igfbp8, Kcnq1 and Tgfβ2) from direct and indirect titin binding partners and non-titin binding partners to validate in protein level with western blotting at three ages (Fig. 5J). We found that proteins Fhl1, Darp, Crp2 and Tgfβ2 were consistent with DNA microarray and QPCR data. Protein Igfbp8 was slightly reduced in d1 Hm which was consistent with DNA microarray data and significantly increased at d20 and 49 that was consistent with both DNA microarray and QPCR data. Protein level of Kcnq1 was not changed at three ages which was consistent with DNA microarray and QPCR data at d1, but not d20 and 49 (Fig. 6J). These data suggest that most of genes validated by QPCR and western blotting have consistency between mRNA level and protein level, and they are consistent with DNA microarray data too, confirming the gene expression changes obtained from DNA microarray data are reliable.
Titin direct and indirect binding partners. The central rectangle depicts the sarcomere positions of different titin segments. The direct titin binding partners are orange ovals; the indirect titin binding partners are yellow rectangles; the direct and indirect titin binding partners with differential expression are red ovals and rectangles. sANK1, small ankyrin 1 isoform; p94, calpain-3; FHL1/2, four-and-a-half-LIM-domain-protein; MyBPC, myosin binding protein C; Nbr1, neighbor-of-BRCA1-gene-1; MURF, muscle-specific-RING-finger-protein; MyHC, myosin-heavy-chain; MyLC, myosin-light-chain; TPM, tropomyosin; CARP, cardiac-ankyrin-repeat-protein; DARP, diabetes-related-ankyrin-repeat-protein; ARPP, ankyrin-repeat-domain-protein-2; TMOD, tropomodulin; MYOT, myotilin; FLNC, Υ-filamin; T-cap, telethonin; minK, potassium-channel subunit; MLP (CRP3), muscle-LIM-protein (cysteine-rich-protein-3); CRP2, muscle-LIM-protein (cysteine-rich-protein-2); ENAH, protein enabled homolog; ANP, atrial natriuretic peptide; BNP, brain natriuretic peptide; KCND2/3, potassium voltage-gated channel subfamily D member 2/3; KCNK2, potassium channel subfamily K member 2; KCNQ1, potassium voltage-gated channel subfamily Q member 1; TNFa-CE (ADAM-17), tumor-necrosisfactor-alpha-converting-enzyme (A-disintegrin-and metalloprotease-17 protein); ADAM33, disintegrin-and metalloprotease-33 protein; IGFBP, insulin growth factor binding protein; ZNF, zinc finger protein; ERK2, extracellular-signal-regulated-kinase-2; ANK2, ankyrin 2; YB-1, Y-box-binding-protein-1; ISOT-3 (USP13), isopeptidase-T-3; USP24, ubiquitin specific peptidase 24; UBC9, SUMO-conjugating enzyme UBC9; UBCH10, SUMO-conjugating enzyme E2C.
We next studied and validated genes involving in calcium release signaling. From our DNA microarray data, we found that genes Trdn, Casq1, Cacna1c, Tnnt1, MyHC involved in calcium handling and sarcomere contractility were differentially expressed. These genes were displayed in schematic diagram of calcium handling and sarcomeric structure (Fig. 6A). We validated calcium handling genes Trdn and Casq1 with fold changes, while other genes (RyR2, Serca2a and phospholamban/PLN) that have no fold changes in calcium handling from DNA microarray. Western blotting analysis demonstrated that Trdn was down-regulated and casq1 was up-regulated in Hm at d1, d20 and d49, while other genes (RyR2, Serca2a and PLB) had no changes (Fig. 6B). These results are consistent with the DNA microarray data. Interestingly, western blot indicated that Trdn and casq1 protein bands had migration difference between Wt and Hm. This could be caused by alternative splicing that is regulated by RBM20 as reported previously . Given that expression profile changes of some calcium handling genes, we assumed that intracellular calcium concentration may be subjected to changes during muscle contraction. Thus, we measured the calcium transient with cardiomyocytes isolated from the heart at d49 using a calcium contractility system (IonOptix). The results indicated that basal level of the intracellular calcium concentration was significant higher in Hm cardiomyocytes than that in Wt (Fig. 6C), while, in response to electrical stimulus, intracellular calcium change was lower in Hm cardiomyocytes than that in Wt (Fig. 6D). However, intracellular calcium decay rate had no significant difference between Wt and Hm cardiomyocytes (Fig. 6E). These data suggest that calcium releasing signaling could be impaired, leading to calcium leaking, while calcium ATPase pump system was physiologically functional and could normally pump calcium from cytosol to sarcoplasmic reticulum (SR) for calcium uptake. Calcium releasing from SR is through a multi-molecular signaling complex channel includeing RyR, Trdn, Junctin and Casq that plays a key role in excitation-contraction coupling (EC-coupling). The calcium enters the cytosol through plasmalemmal voltage-gated calcium channels and activates RyR channel to release more calcium from SR for muscle contraction [66-68]. However, the RyR channel is also positively controlled by the luminal side of the channel [66, 69-70]. Auxiliary proteins (Trdn, Junctin and Casq) with luminal localization mediate the effect of luminal calcium on RyR [71-72]. Casq is a major calcium binding protein and changes conformation upon calcium binding, resulting in interaction with RyR to sense luminal calcium [73-76]. Trdn and Junctin mediate the inhibitory interactions between Casq and the RyR channel . Therefore, down-regulated Trdn and up-regulated Casq1 may cooperate for calcium releasing. Since RyR2 is not changed based on our data, it still remains unknown how calcium leaking occurs in Rbm20 Hm rats. Previous study indicated that RBM20 regulates alternative splicing of RyR2, leading to a 24bp insertion in Rbm20 Hm rats , which could be a cause for calcium leaking. However, further study is needed to confirm this hypothesis. Nevertheless, expression changes of Trdn and Casq1 may play a role in the impaired calcium release signaling in Rbm20 Hm rats. On the other hand, SR calcium uptake is mediated by a calcium ATPase Serca2, whose activity is reversibly regulated by PLN . Since no changes of these two proteins were found in Rbm20 Hm rats, calcium uptake appears normal, which was consistent with the measurement of intracellular calcium decay rate (Fig. 6E).
Validation of differentially expressed genes with QPCR and western blot. (A, B and C), Expression level of selected titin direct binding partners with DNA microarray and QPCR; (D, E and F), Expression level of selected titin indirect binding partners with DNA microarray and QPCR; (G, H and I), Expression level of selected genes that are not titin binding partners with DNA microarray and QPCR; (J), Validation of selected proteins from titin direct and indirect partners and non-titin binding partners with western blot. D, day; Wt, wild type; Hm, homozygous Rbm20 knockout; GAPDH, protein loading control. Mean±SEM (n=3); *P<0.05; **P<0.01; ***P<0.001; #P<0.05; ##P<0.01; ###P<0.001.
Transcriptional network of calcium handling genes and intracellular calcium changes. (A), Schematic diagram of calcium release signaling and sarcomere; (B), validation of key calcium handling proteins with western blotting; (C), Intracellular calcium concentration changes with cardiomyocytes isolated from the rat heart at d49. Resting FFI, basal intracellular calcium level; (D), ΔFFI, Rise in intracellular calcium in response to electrical stimulus shown as changes in Fura-2 fluorescence intensity; (E), D, day; Wt, wild type; Hm, Rbm20 homozygous knockout; GAPDH, protein loading control. Mean ± SEM (n=30), *P < 0.05, **P<0.01.
Our results reveal that the muscle tissue specific splicing factor RBM20 not only regulates gene alternative splicing but also gene expression in the heart. Previous studies have identified approximately thirty genes that are undergoing alternative splicing regulated by RBM20. In this study, we have demonstrated that RBM20 also modulates gene expression of about four hundred genes with heart maturation. Intriguingly, we found that for certain genes such as Trdn and MyHC, RBM20 regulates their splicing as well as their expression level. Rbm20 deficiency in animal models leads to dilated cardiomyopathy and heart failure, and mutations found in RBM20 are also associated with dilated cardiomyopathy in human patients. However, the detailed mechanisms of how RBM20 causes heart dysfunction remain unclear. Our data suggest that the mechanisms of both gene splicing and gene expression regulated by RBM20 will require more detailed study in the future. Particularly, whether gene expression is directly regulated by RBM20 or is a secondary effect remains unknown. At least, previous studies have shown that pre-mRNA splicing can be coupled to transcription and crosstalk and impact each other [78-79], and trans- and cis-acting factors can regulate RNA polymerase II elongation [80, 81]. However, whether RBM20-mediated transcription is involved in coupling to RBM20-mediated splicing is very interesting to be further studied. In conclusion, our results provide new insights into the mechanism of RBM20-induced heart disease.
RBM20: RNA binding motif 20; RRM: RNA-recognition domain; SR: serine /arginine; E16: 16 days fetuses; SDS-VAGE: sodium dodecyl sulfate agarose gel electrophoresis; Wt: wild type; Hm: homozygous knockout; RMA: robust multi-array analysis; LV: left ventricule; FFI: Fura-2 fluorescence intensity; d: day; yr: year; GO: Gene Ontology; GSEA: Gene Set Enrichment Analysis; Fhl1: four-and-a-half Lim domain protein-1; MARPs: muscle-ankyrin-repeat proteins; SR: sarcoplasmic reticulum; EC-coupling: excitation-contraction coupling; sANK1: small ankyrin 1 isoform; p94: calpain-3; MyBPC: myosin binding protein C; Nbr1: neighbor-of-BRCA1-gene-1; MURF: muscle-specific-RING-finger-protein; MyHC: myosin-heavy-chain; MyLC: myosin-light-chain; TPM: tropomyosin; CARP: cardiac-ankyrin-repeat-protein; DARP: diabetes-related-ankyrin-repeat-protein; ARPP: ankyrin-repeat-domain-protein-2; TMOD: tropomodulin; MYOT: myotilin; FLNC: Υ-filamin; T-cap: telethonin; mink: potassium-channel subunit; MLP (CRP3): muscle-LIM-protein (cysteine-rich-protein-3); CRP2: muscle-LIM-protein (cysteine-rich-protein-2); ENAH: protein enabled homolog; ANP: atrial natriuretic peptide; BNP: brain natriuretic peptide; KCND2/3: potassium voltage-gated channel subfamily D member 2/3; KCNK2: potassium channel subfamily K member 2; KCNQ1: potassium voltage-gated channel subfamily Q member 1; TNFa-CE (ADAM-17): tumor-necrosisfactor-alpha-converting-enzyme (A-disintegrin-and metalloprotease-17 protein); ADAM33: disintegrin-and metalloprotease-33 protein; IGFBP: insulin growth factor binding protein; ZNF: zinc finger protein; ERK2: extracellular-signal-regulated-kinase-2; ANK2: ankyrin 2; YB-1: Y-box-binding-protein-1; ISOT-3 (USP13): isopeptidase-T-3; USP24: ubiquitin specific peptidase 24; UBC9: SUMO-conjugating enzyme UBC9; UBCH10: SUMO-conjugating enzyme E2C.
This work was supported by the National Institute of Health/National Institute of General Medical Sciences [NIGMSP20GM103432]; the BGIA from the American Heart Association [16BGIA27790136 to WG]; the USDA National Institute of Food and Agriculture [Hatch project 1009266 to WG] and the National Institutes of Health [grant HL77196 to MLG]. The authors are grateful to Mark D. Hanna for his technical assistance, animal care and laboratory management.
W.G., and M.G., conceived and designed the experiments; Z.C., Y.Z., W.Q., S.M., C.H. and W.G. performed the experiments and interpreted the data; W.G. wrote the paper; M.G., revised the paper.


Domino's Pizza has been too confrontational with its biggest franchisees, says one of its major investors.
The spat started last year when powerful franchisees claimed they were not receiving a big enough slice of the delivery firm's profits – leading to them threatening to halt new store openings.
Philip Macartney, at Columbia Threadneedle, a top ten shareholder, said: 'I have concerns with the way [Domino's] has dealt with that relationship. It's become adversarial between the franchisees and the company. It was a failing not to understand the franchisees' concerns and pressures.
The fund manager also turned his ire on the franchisees, claiming they had been 'over-earning a few years ago'.
Domino's share price has fallen 36 per cent since June. The board – which faces angry shareholders at their annual meeting a week on Thursday – is seeking a replacement for chief executive David Wild who lost three finance chiefs in as many years.
A Domino's spokesperson said: 'We have an open and ongoing dialogue with our franchisees and are actively exploring win-win solutions, which will incentivise growth and benefit them, the plc and most importantly, customers.


Most research suggests that retirees are very significant contributors to Australian charities, and indeed contribute more than any other age group - which makes sense from both the community and financial perspective. The chart below, sourced from NAB's Charitable Giving Index Report of February 2018, illustrates just how charitable giving generally increases with age.
The downside is that many retirees are the focus of incessant charity marketing, by telephone, door-to-door callers and by charity marketers in shopping centres. Many of these activities are carried out by professional marketing companies, and many individuals provide donations without being aware that a substantial proportion of the funds collected are paid to the marketing company, to individual collectors and indeed shopping centres that charge access fees to collectors. Presently, only in NSW and Victoria is it required that face-to-face collectors who door knock or collect in shopping centres display badges which indicate they are "paid" volunteers. If unclear, ask the collector directly about their status.
Many charities take the view that individuals will not donate without being first "asked to give", and that collecting any money, even if only 50% or less of the amount collected, means that they still receive money they would not otherwise have generated.We don't concur and believe that raising money in this fashion will eventually undermine the public's confidence in charities in Australia and that it already leads to situations where individuals are unfairly pressured to make contributions.
Not participate in offers by charities to assist you in drafting a will. Regardless of whether they explicitly or implicitly request a "bequest or legacy" you should not have any potential beneficiary involved in the drafting of a will; we believe there is an innate conflict of interest. The drafting and maintenance of a will requires unbiased professional advice and we strongly recommend the involvement of a solicitor whom you appoint and remunerate.
Support calls for charities to be subject to the "Do Not Call Register". A recent Choice report indicated that calls from charities, or commercial marketing companies that represent them, were the single largest source of unsolicited telephone calls - with more than 25% of Australians interviewed saying that they had received unwanted calls from a charity each week.
In other words, by all means continue to donate to charities, but seek to do it in a more targeted and effective fashion with less of your donation money going to commercial fundraisers.


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text:
Foreign Relations of the United States, 1977–1980, Volume III, Foreign Economic Policy
148. Minutes of the Bonn Economic Summit Meeting1
Bonn, July 17, 1978
BONN SUMMIT
Schmidt: Let's now turn to the declaration.
Giscard: (to Schmidt)—Regarding the growth paragraph, I would hope that you could add some additional language of greater specificity on growth. If possible, it would give greater strength to the text. Perhaps indicate anticipated growth increase of ½–1% of GNP.
Schmidt: We would indicate a willingness to say this but others had criticized mention of this figure.
Carter: What is the most exciting figure, the highest figure, you would be willing to mention?
Schmidt: I would be willing to say ½% to 1% increase in expenditure. But your delegation turned this down.
Carter: I agree with up to 1%.
Callaghan: I am sure that if the phrase is left in a general way, we will be asked what was said in the meeting. I therefore agree with Giscard and Carter.
Schmidt: The U.S. Delegation requested the figure be taken out. Do I now understand that the U.S. President has changed the mind of the U.S. Delegation?
Carter: About how much is 1%?
Schmidt: 13 billion deutsche marks.
Giscard: Yesterday Fukuda used the figure of 7% growth. Since others are using figures in their paragraphs, we might remind people in the declaration that the Japanese target is 7%.
Fukuda: Others have not mentioned specific growth targets. Therefore it is not appropriate for Japan to give specific figures. The Japanese people may feel that Japan has been singled out. The word "significantly higher" implies 7%. If asked, you can say 7% growth target is what was meant. You can quote me.
Trudeau: If it helps in this exercise, I can include for Canada a figure of up to 5%.
Giscard: Now that we have a Canadian figure, we need a Japanese figure.
Fukuda: But that is not a figure which represents a growth target for the economy. If Japan is the only one to give a growth target, I can't do it. You can tell people that the Japanese Prime Minister has used the figure of 7%.
Carter: Would you be willing to set a percentage of increase that you will set as a goal?
Fukuda: Last year our growth was 5.4%.
Carter: Would you be willing to indicate a net increase in growth of about 1.5%.
Fukuda: The present phrase is all right. If we set a target, we will be the only country to do so. People will add up 5.4% and 1.5% and it would be almost the same thing as setting a target.
Schmidt: Canada has indicated its intention to achieve a 5% growth rate in 1978. Germany has said it will take additional measures to increase growth of up to 1% of GNP. Giscard said France will increase its budget deficit by ½% of GNP. Italy has indicated that it will do 1.5% better than in 1978. In light of the foregoing four countries which have made quantitative pledges, it would be useful if Japan were to do likewise. Carter has used the phrase "which is 1.5% higher than the previous year." This would not single you out. It would be similar to the foregoing four countries.
Fukuda: In the case of the U.S., it did not spell out its growth rate. There would be certain criticisms within Japan when we are asked questions of why we said in the declaration 7% growth.
Schmidt: Maybe we should look for another kind of language which avoids mentioning the growth rate such as "measures toward the expansion of domestic demand on the order of 1.5% of GNP.
Fukuda: Last year we had 5.5% growth. If you add 1.5, you will be setting a target in a different way. We will not oppose the other countries' paragraphs, which are somewhat vague. We can say that we will do higher than last year's 5.5%. We will obtain our real growth target for FY '78. This means 7%. It is quite clear that for FY '78 our target is announced. This satisfies your intention, doesn't it?
Schmidt: Could we say Japan will achieve its real growth target as already announced for FY '78?
Fukuda: I could agree to buy Carter's suggestion that we indicate we will do 1.5 above the previous year.
Callaghan: The press is likely to say what does this all add up to on unemployment and growth.
Carter: We can say that we all have made several strides forward. Germany and Japan agreed to greater growth and we agreed to restrict oil consumption and inflation. It is a brave new world.
Schmidt: We cannot calculate the effect of these measures. We cannot foretell the results; the situation looks a little brighter.
Giscard: A slightly more scientific answer is needed. The origin of this effort was in the OECD Working Group, under Charlie Schultze, working with the European Commission. The OECD indicated that if growth were 4.5% there would be a certain fall in unemployment because 4.2% seems to be the threshold in Europe above which unemployment declines. All figures for the Schultze group except those for the FRG seem to have been taken up here. We will ask the OECD to assess the impact. But the net results should enable us to pursue an increased growth rate and a decrease in unemployment.
Schmidt: There seems also to be some issue of subsidies and discipline.
Carter: We think we should say we will look for greater discipline in the use of subsidies so that they do not distort the normal flows of world trade. We would like to add a sentence to this effect.
Callaghan: It depends on how we interpret this. I am not ready to see firms destroyed by world recession when they require subsidies to keep them going.
Jenkins: Suddenly to insert this at this stage when this is a complex MTN issue is very difficult.
Giscard: Countries using subsidies feel they do not distort world trade. We do not believe this sentence would have practical content; in fact we believe it will have no meaning.
Schmidt: I see this as an instrument for countries to avoid pressures for new subsidies. I do not think it is being pointed against participants here.
Callaghan: I don't particularly want this wording at all.
Schmidt: The UK has not been mentioned.
Callaghan: If anything would be mentioned, it would be the common agricultural policy.
Giscard: It is a question of method. We can't open the door on subsidies at 4:30. We can accept this because it has no practical consequences. But this would not be honest on our part. U.S. firms for instance have favorable contracts on defense production. Is this a subsidy? Let's put this on the agenda for the next Summit.
Carter: I will yield on this issue but I do not accept the French argument. We are concerned about the excessive use of subsidies. We tried in our phrasing to be as innocuous as possible but apparently this caused some embarrassment to some countries. We will not insist on it, but we are concerned about excessive and perhaps growing subsidies.
Schmidt: We must not [now] spend some time on North/South issues.
Fukuda: We are surrounded by LDCs. We enjoy a close relationship with ASEAN, whose views and philosophy we find compatible with industrialized nations. The developing countries want us to suppress protectionism and there are a number of institutional matters on which the developing countries request our decisions. They want expanded imports by the developed countries. They also want establishment of a common fund and want specific consideration by developed countries of commodities. They urgently want a conclusive attitude on the common fund to achieve results by next year's UNCTAD meeting in Manila.2 It is important for the developed countries to take some improved philosophy or posture. On the common fund, we should show an attitude of determination to work on the common fund idea to provide a conclusive idea by the end of the year. This would be a meaningful gesture to the LDCs.
As for Japan, we will double aid in 3 years. We are also trying to improve our growth and to provide co-financing through the international financial institutions. Some success resulted from the UNCTAD meeting last March,3 but we must still deal with the mounting debt problem of LDCs and develop a conclusive attitude on rising debt.
Schmidt: In addition to the general evaluation of the situation, do you want to alter the communique?
Fukuda: I do not propose any changes, but you mentioned common fund. In addition to the draft, I suggest that we approach our [Page 470]experts to come up with conclusions by the end of the year. The communique is, however, okay in draft.
Andreotti: I am not proposing any changes, but I have two comments. It is important that in addition to concerning ourselves with the problems of the developed countries we also concern ourselves with the problems of the developing countries. I was impressed with what Prime Minister Fukuda said about the problems of the future, such as the water and arable land. We should study in the future our approach to the LDCs. It is important to remind people in the developed countries, particularly our young people, of the problems of the developing countries—to remind them that 1.5 billion people in this world earn less than $90 a year. Maybe it will make us more willing to sacrifice in our own countries if we do not forget world poverty.
Carter: I would like to add on the common fund point in the communique that we should pursue negotiations to a successful conclusion. We should get going on the common fund and make clear that we are going to go ahead and wrap it up.
Giscard: There is considerable emphasis on multilateral aid in this paper. It is also important that we give attention to bilateral aid for political reasons. We should highlight our own aid to indicate the progress we have made in dealing with the problems of the LDCs.
Schmidt: I see no problem adding Carter's four words. However, I support Giscard on aid. Most people in LDCs have no idea of where the aid they get comes from when it comes from the World Bank. It is also difficult to raise appreciation for foreign aid when LDCs criticize my country. LDCs should not see aid as coming out of the vaults of the World Bank. Aid depends on national legislatures. What Giscard said was important; not too much aid should be multilateral. We should make it clear to LDCs where the aid comes from.
With respect to the Eastern countries, I do not agree that we should say "invite." We invited them at London to provide more aid and nothing happened. Instead, they stepped up their deliveries of weapons and advisers to Africa—including military advisers from the GDR. Invite is meaningless. We should spell out that the 7 countries here reproach COMECON for not contributing substantially to LDCs.
The last section of this declaration is meaningless on commodities. There are no direct German interests involved. There are no Germanophone countries. I am talking from a general economic point of view. I am concerned about and question whether the common fund is essential, as well as certain commodity agreements. To my mind, stability of export earnings is even more important for LDCs. A copper agreement would be good for the Soviet Union and give it a lot of profits. But a common fund should not enable the rich to get richer. A copper agreement would help Zaire, Zambia and Chile, but it would be better for [Page 471]the U.S. and the Soviet Union. Commodity agreements are of more use to developed than to developing countries. I don't think my consumers and taxpayers should be asked to contribute to the well-being of large developed countries rather than poor LDCs.
I am extremely dissatisfied with the way in which examination of stabilization of export earnings has been handled so far. The common fund and commodity agreements mean large rich countries get more benefits than poor countries. The great majority of LDCs derive no benefit whatsoever. This is what I feel. We will say this in international fora about this approach.
I also repeat that I find the language on COMECON too friendly.
Trudeau: You speak from the heart, but you also speak for a lot of us.
Carter: We should say we deeply regret the failure of COMECON to assist developing countries.
Trudeau: On the point above, the common fund and STABEX, we are all growing impatient with the failure to find methods to achieve this objective. We all want to transfer resources to LDCs. But when we gave lots of bilateral aid there was a great deal of overlap and confusion.
On the common fund, LDCs want to stabilize prices and price increases. If third world countries rely on one commodity, they face very real problems. People may use less sisal, but we can't be blamed for that. We should use the time between now and the next Summit to clarify our objectives and not mix in the common fund, Lome, etc. The 774 always try to do everything at the same instant. The LDCs want something because it sounds good.
Callaghan: I support Pierre's point about the risk of countries going from multilateral to bilateral aid. We can influence governments a bit when aid is bilateral but very few changes are made as a result. The common fund idea has changed a lot since 1974 and 1975. It now has become a political necessity.
Schmidt: I acknowledge that although it will not help 90% of LDCs.
Callaghan: We must maintain our position on the common fund until the developing countries move, but when they do, it would be advisable for us to support the second window. If the price is to give in on the second window and some direct contributions—if that is required to get it out of the way, we should move at an appropriate moment.
I would like to see agreement to double the capital of the World Bank, which is $31 billion now. The World Bank handles its affairs well. I would like to see the World Bank used as the main instrument for the development process. I hope we can return to this. Also, the level of aid as a percentage of GNP has substantially declined. This is a bad sign.
On retroactive terms adjustment, we have reduced debt. Let's face it, the LDCs will never repay their loans. It would have some meaning in LDC eyes if we move more in this direction.
Schmidt: This is the first time we are discussing the LDC issue. You should feel free to put up new ideas on this new subject.
Giscard: At the next meeting we should give more time to North/South policy. Regarding the paragraph on COMECON, we should say we invited once more COMECON to do their share in assisting LDCs.
Carter: We should say we deeply regret the failure of COMECON countries to do their share of financial assistance to LDCs and invite them once more to do so.
Schmidt: I have a proposal to make outside of the text—that is a joint procedure among ourselves envisaging further action. Our notetakers will bring to our attention proposals made in this field. We should evaluate our situation in the field of development aid. In less than two years the developing countries will become aware that the common fund would not really help them. We should prepare for this.
I should also say that nothing has been done on the study of export earnings since our meeting at Rambouillet.
Trudeau: We should say that we will meet before the next Summit to tackle this problem. Were you counting on our aides to assess progress in the next 6 months?
Schmidt: Yes. They should assess progress across the board.
Carter: I would like to propose that we meet in Japan before the end of June next year.
Schmidt: Our notetakers and aides should follow up our discussion.
Giscard: We should say that our officials will meet toward the end of the year to review progress. We should not announce a date for another Summit because the economic situation may make a meeting necessary at one time or another. We should say that the next meeting will take place as and when circumstances make it desirable.
Fukuda: Followup is an important matter. I would like to urge on you that we say we will followup in an appropriate phrase in the communique.
Schmidt: I agree. We should also say we have instructed our Ministers to, by the end of '78, convene in order to review progress in car[Page 473]rying out the provisions of this declaration and that we intend also in 1979 to have a similar meeting between ourselves.
Giscard: We have not said this in the past. We do not want to institutionalize this. There are two advantages of announcing our intention to come together in the spring of '79. If we announce today, people will not think that such a meeting when called is a signal of urgent danger. And there will not be such great expectations.
Callaghan: I agree. I would like to institutionalize this body so that it meets every twelve months. It also reduces pressures from others to get in.
Giscard: Let's not be bureaucratic. Let's keep some flexibility and say that we have decided to meet again when the situation seems appropriate. We can say we reflected on the invitation of Japan to meet next year. In the declaration we can say we envisage a similar meeting between ourselves at an appropriate time next year. We should also say that we instructed our representatives to, by the end of 1978, convene in order to review progress in carrying out this declaration.
Schmidt: I think this was an excellent meeting. Let's now adjourn and then we can go over to the press building.5
Source: Department of State, Office of the Secretariat Staff, Records of Cyrus Vance, Secretary of State, 1977–1980, Lot 84D241, Box 9, Vance NODIS Memcons 1978. Secret. Drafted on August 9. For more information on the drafting of these minutes and a list of Summit participants, see footnote 1, Document 145. This fourth session of the Summit, which took place in the Palais Schaumberg, began at 3:34 p.m. and ended at 6:18 p.m. (Carter Library, Presidential Materials, President's Daily Diary)↩
UNCTAD V took place in Manila May 7–June 3, 1979.↩
The negotiating conference on the Common Fund, agreed to at UNCTAD IV in May 1976, held a preliminary meeting in Geneva March 6–April 3, 1977.↩
Reference is to the Group of 77, or G–77, the developing countries group established at the conclusion of the first UN Conference on Trade and Development in 1964. For the Lomé Convention, see footnote 6, Document 24.↩
For the final text of the Bonn G–7 Summit Declaration issued on July 17, see Public Papers of the Presidents of the United States: Jimmy Carter, 1978, Book II, pp. 1310–1315. Carter's remarks to the press at the conclusion of the Summit are ibid., pp. 1309–1310.↩
Foreign Economic Policy
International Monetary and Trade Policy (Documents 1-252)
North-South Issues; Commodities Policy (Documents 253-356)
Andreotti, GiulioCallaghan, JamesCarter, James Earl (Jimmy)Fukuda, TakeoJenkins, RoySchmidt, HelmutSchultze, Charles L.Vance, Cyrus R.
ASEANCOMECONFRGFYGDRGNPGroup of 77G–7G–77LDCMTNOECDSTABEXUKUNUNCTAD


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The Evening Standard City Editor on the end of Nicoli's vision for Emi, and Sets
By Anthony Hilton for MailOnline
Updated: 19:00 EST, 4 October 2000
Eric Nicoli, the boss of EMI, today called time on his attempt to sell the business to the American Time Warner group in the face of implacable opposition from the regulators in Brussels. Shareholders should rejoice. This was a bad deal made worse by the apparent willingness of Nicoli to dismember the company and sell its best assets in an attempt to appease the regulators.
Those tempted to support Nicoli's vision of the future should look at his earlier career at United Biscuits. For 21 years United Biscuits owned Keebler, a US cookie manufacturer but the business hit problems in the 1990s when it went head-to-head with Pepsi in snacks. UB then decided to get out and in the closing days of 1995 sold it for under $600m.
The people who bought Keebler then simply withdrew from snacks, refocused the business and made it highly profitable - by using commonsense rather than rocket science. Coincidentally they have now put it up for sale again and with both Kellogg and Campbell Soups said to be interested, Wall Street analysts say it could fetch $4,500m. If it does that it will be seven times as much as UB got in less than five years.
The guy who handled the sale and indeed the strategy that got Keebler into the mire in the first place was Eric Nicoli, the same who is now chairman of EMI.
Everyone can make mistakes but I do wonder if executives in Britain are becoming too willing to sell out rather than knuckle down and tackle difficult management problems. It is all very well loading executives up with share options but it does perhaps give them an incentive to opt for short-term easy answers cloaked as the pursuit of shareholder value.
Sets lessons
One of the issues thrown up by the current malaise of the Stock Exchange is the downside as well as the upside of outsourcing. About seven years ago, after it had lost a mint on a new and over-ambitious settlement system, the Exchange outsourced its IT department to Andersen Consulting.
That firm designed and built the electronic trading platform Sets and delivered the savings that allowed the Exchange to claw its way back into profit. At the time, it seemed all gain for the Exchange. It got a state-of-the-art system it could not otherwise have afforded, with none of the risk of building and development, and it got a share of the cost savings going forward. But now it appears to be not such a oneway deal. Indeed, it should give pause for thought for all those in the financial community, under pressure to outsource everything that is not a core competence.
The Exchange's mistake was to fail to recognise how crucial technology was to its future. The Exchange did not secure the rights to Sets so, using the knowledge gained in London, Andersen got the contract to design and build Xetra, the trading platform for the Frankfurt Stock Exchange. It therefore quite legitimately delivered to London's main Continental competitor a later and superior system. Nor was that all. The original outsourcing contract failed to make sufficient provision for future upgrades with the result that there is no natural successor to Sets. That is why, looking to life after Sets, the Exchange is looking into a technological black hole.
The easy moral is that no organisation should outsource anything that is crucial to future competitiveness, but no one realised that that was what it was doing - and besides, being broke, the Exchange had little choice. When it went for Sets, trading technology was not seen as crucial. Only with hindsight did it become apparent that technology was the future.
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Chris Matthews is the host of Hardball (MSNBC) and The Chris Matthew Show (NBC). He was a presidential speechwriter for the Carter administration and a top aide to former Speaker of the House, Thomas P. "Tip" O'Neil.
In his 2001 book, Now, Let Me Tell You What I Really Think, Chris Matthews exposes the horrors of bullfighting.
The United States has its ballparks. Spain has its bullrings. Here, major league cities sport stadiums that seat tens of thousands of baseball fans, while minor league cities have their own, smaller ball yards.
Go to a city like Seville and you'll see a 10,000-seat bullring. Head south into the heart of Andalusia and you'll reach Ronda, with its 5,000-seat plaza de toros.
Ronda is recognized as the birthplace of bullfighting in Spain, and its elegant showplace opened in 1785. It drew the likes of Ernest Hemingway and Orson Welles, who found fascination in what Spaniards call the "equestrian art" of bullfighting.
Letter Written Regarding a Catholic High School's Field Trip to a Bullfight.
When I read that your Catholic (that is still a Christian faith isn't it?) High School students' trip to Spain included their seeing and supporting a bullfight I was outraged! The Catholic Church has worked hard to shed its scandals of late, but then your staff members use such gross judgment in allowing impressionable teenagers to view the cruel "blood sport" of bullfighting on their trip. How does a person of faith actually justify that in their mind? What are you thinking? Are you telling me that they actually have to physically support this event to understand this ugly/dark part of the Spanish culture? Couldn't you have just shown them some SHARK (animal rights group protecting livestock) videos that are very graphic in its proof of the cruelties inflicted on livestock, particularly those used for entertainment by the greedy and barbaric?
Michael Ogorzaly, who died at age 58 on October 14, 2006, suffered a broken neck as a college student, when a car in which he was a passenger was involved in an accident. Confined to a wheelchair thereafter, Ogorzaly completed his education and went on to teach Spanish and Latin American history at Chicago State University. When Bulls Cry was his second book, addressing a topic which had become one of his focal concerns.
De-romanticising the bullfight spectacle with a dose of anguishing realism in chapter one, Ogorzaly goes into the history behind it. Chapter two discusses thegeneology of bullfighting, revealing that the present day corrida, which originated in the 18th century, has very little connection with Spanish tradition.


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text:
Read to Achieve the Magic!
Discovering Magic Pearls
The Peanut Butter Trap, Hate...
Kirkus Indie Professional Review for: The Hunt for the Magic Pearl
"... this book is classroom ready, as well as a worthy bedtime tale." --Kirkus Review
Buy Me Now
"Well, where are they?" StarFire demanded. "Where is that oyster and, where is that Magic Pearl?" Where were they indeed? Had SeaStar hidden them to keep the pearl safe?
Mermaid glamour meets action and suspense in Perry-Church's vividly imagined debut fantasy book for children.
Nestled deep in an ancient ocean, the kingdom of Merland is a formidable and beautiful place in which to grow up. It's also the home of two mermaids named Shimmer and StarFire, as well as their tag-along little sister, SeaStar. After their mother's death in a shark attach, they're a little wary of wandering alone. However, the tantalizing promise of a magic pearl lures them out on a treasure hunt in the open sea, where they're sure to find both danger and adventure. On their way, one of the mermaids also finds a surprising source of strength. The story is set against the backdrop of a richly imagined undersea kingdom; for example, the author details the dangers of Merland as well as some of its strange, magical elements. However, the story also offers plenty that young readers will relate to, including bossy older siblings and struggles with self-image and self-esteem. The book touches on deeper issues of loss and grief, and handles the death of the young mermaids' mother with sensitivity and tact. The timid, awkward-looking SeaStar is an endearingly unlikely heroine, and readers will be surprised and delighted to see their experiences reflected in hers. With its glossary and several pages of mermaid-related information, this book is classroom-ready, as well as a worthy bedtime tale. Some members of the book's target audience may find that its large amount of text and somewhat advanced vocabulary ("she had a streamlined figure with pronounced human features") make it a challenging independent read. However, it promises to enchant many others with its equal measures of magic, warmth, and thrills.
A fun, exciting underwater romp.
--Kirkus Reviews
Kirkus Indie, Kirkus Media LLC, 6411 Burleson Rd., Austin, TX 78744
indie@kirkusreviews.com


At his memorial in Washington DC, the statue of Martin Luther King is half finished. The legs and back disappear, like a ghost, into a slab of uncarved white marble. His face is determined and resolute, but not yet satisfied. A line from his "I Have a Dream" speech, from 1963, is etched into the rock: "Out of the mountain of despair, a stone of hope." His sculpture is unfinished for a reason – the struggle continues.
The civil rights movement was about racial equality, but it was also about living up to the guarantees of the constitution, to "cash a cheque," as Dr King put it, "[on the] promissory note to which every American was to fall heir" – that all individuals, regardless of who they are or where they're from, would be given the unalienable rights of life, liberty and the pursuit of happiness.
Now something's brewing again. Fuelled by the far right rhetoric of the Trump presidency, and a growing distrust in political and economic systems, a new civil rights movement has been born – one based not just on race but on LGBQT rights, economic rights, immigration rights and the environmental rights of all future generations. The new civil rights movement, the resistance as it has come to be known, may be angry at Trump, but it's also about cashing that cheque.
Celebration of black lives: the Smithsonian National Museum of African American History and Culture.
Which is why I've come to Washington DC – to see where the great experiment in self-governance began. There is nowhere better to understand America's political hopes and dreams, to get a sense of the roots of the division in America and the future of the resistance now.
But there are other reasons to visit, too. Zagat recently rated Washington DC as the number one foodie destination in America. And the historic Georgetown district, with its grand, red-brick homes and cobbled pavements, is lively and fun, filled with shops, restaurants and bars.
There are many decently priced hotels in Washington. One of the best value is probably the Tabard Inn in Dupont Circle, mentioned in John Grisham's Pelican Brief. But if you can afford to splash out for one night the Watergate Hotel is worth it for its legacy alone. It was here, in 1972, that employees of President Nixon spent the night after illegally raiding the offices of the Democratic National Committee nearby. It may have been marred by the Watergate scandal, but it's spectacular: bold, midcentury modern design filled with gold, glamour, and the best whisky bar in the city.
If you're here for the history, the best place to start is where Dr King ended his famous march, on the steps of the Lincoln Memorial. He chose this spot because, in many ways, it represented a full circle for African Americans. The man who had signed the Emancipation Proclamation and freed the slaves stood watch as thousands came to demand the fruits of that promise. It's a moving place. The great obelisk of the Washington Memorial, the pillars of the National Archives, home of the Constitution and the Bill of Rights, and the rotunda of the Capitol – they are a powerful sight, but a conflicting one, too.
Many of these monuments were made by slaves. Parts of the Capitol building, the Statue of Freedom which sits on top, even the White House itself were built by men in chains. This country was founded on that paradox. Lincoln freed the slaves and was shot in Ford's Theatre down the road for doing so. Dr King's dream, those words that seem so obvious now, were viewed by many as a threat: J Edgar Hoover, the director of the FBI at the time, worked tirelessly to undermine his efforts. The Civil Rights Act, far from being unanimously received, was constantly obstructed by southern members of the congress. Only the assassination of Kennedy, and the outrage that followed, had the political muscle to push it through.
Away from the politics, there is much else in Washington to admire. Ben's Chili Bowl on U-Street, home of the chili half smoke, is a DC institution and has welcomed everyone from Chris Rock to the cast of House of Cards. There's also Capitol Lounge for pizza and $5 margaritas; and the Cozy Den Coffee Shop, with the excellent bookstore above which hosts regular author talks and events. Baba, just across the Potomac river in Arlington, serves craft cocktails in a cool speakeasy setting and a chocolate and orange dessert that tastes like Jaffa Cakes.
I saved the best until last with a visit to the Smithsonian National Museum of African American History and Culture. The first museum of its kind in the country, it has sold out every day since it opened a year ago. The journey begins in the basement, with slave ships coming to the New World, and winds slowly upwards through cotton fields and segregation to three floors of art, music, sports, dancing – a celebration of African American culture and the influence of black lives on the country today. Nowhere is the story of America's division, and the bridges that have overcome it, better told. What better way to end a trip in Washington?


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