Rbm20 regulates titin alternative splicing as a splicing repressor

Titin, a sarcomeric protein expressed primarily in striated muscles, is responsible for maintaining the structure and biomechanical properties of muscle cells. Cardiac titin undergoes developmental size reduction from 3.7 megadaltons in neonates to primarily 2.97 megadaltons in the adult. This size reduction results from gradually increased exon skipping between exons 50 and 219 of titin mRNA. Our previous study reported that Rbm20 is the splicing factor responsible for this process. In this work, we investigated its molecular mechanism. We demonstrate that Rbm20 mediates exon skipping by binding to titin pre-mRNA to repress the splicing of some regions; the exons/introns in these Rbm20-repressed regions are ultimately skipped. Rbm20 was also found to mediate intron retention and exon shuffling. The two Rbm20 speckles found in nuclei from muscle tissues were identified as aggregates of Rbm20 protein on the partially processed titin pre-mRNAs. Cooperative repression and alternative 3′ splice site selection were found to be used by Rbm20 to skip different subsets of titin exons, and the splicing pathway selected depended on the ratio of Rbm20 to other splicing factors that vary with tissue type and developmental age.     

Removal of immunoglobulin-like domains from titin’s spring segment alters titin splicing in mouse skeletal muscle and causes myopathy

Titin is a molecular spring that determines the passive stiffness of muscle cells. Changes in titin’s stiffness occur in various myopathies, but whether these are a cause or an effect of the disease is unknown. We studied a novel mouse model in which titin’s stiffness was slightly increased by deleting nine immunoglobulin (Ig)-like domains from titin’s constitutively expressed proximal tandem Ig segment (IG KO). KO mice displayed mild kyphosis, a phenotype commonly associated with skeletal muscle myopathy. Slow muscles were atrophic with alterations in myosin isoform expression; functional studies in soleus muscle revealed a reduced specific twitch force. Exon expression analysis showed that KO mice underwent additional changes in titin splicing to yield smaller than expected titin isoforms that were much stiffer than expected. Additionally, splicing occurred in the PEVK region of titin, a finding confirmed at the protein level. The titin-binding protein Ankrd1 was highly increased in the IG KO, but this did not play a role in generating small titin isoforms because titin expression was unaltered in IG KO mice crossed with Ankrd1-deficient mice. In contrast, the splicing factor RBM20 (RNA-binding motif 20) was also significantly increased in IG KO mice, and additional differential splicing was reversed in IG KO mice crossed with a mouse with reduced RBM20 activity. Thus, increasing titin’s stiffness triggers pathological changes in skeletal muscle, with an important role played by RBM20.     

Altered Contractility of Skeletal Muscle in Mice Deficient in Titin’s M-Band Region

We investigated the contractile phenotype of skeletal muscle deficient in exons MEx1 and MEx2 (KO) of the titin M-band by using the cre-lox recombination system and a multidisciplinary physiological approach to study skeletal muscle contractile performance. At a maximal tetanic stimulation frequency, intact KO extensor digitorum longus muscle was able to produce wild-type levels of force. However, at submaximal stimulation frequency, force was reduced in KO mice, giving rise to a rightward shift of the force-frequency curve. This rightward shift of the force-frequency curve could not be explained by altered sarcoplasmic reticulum Ca²⁺ handling, as indicated by analysis of Ca²⁺ transients in intact myofibers and expression of Ca²⁺-handling proteins, but can be explained by the reduced myofilament Ca²⁺ sensitivity of force generation that we found. Western blotting experiments suggested that the excision of titin exons MEx1 and MEx2 did not result in major changes in expression of titin M-band binding proteins or phosphorylation level of the thin-filament regulatory proteins, but rather in a shift toward expression of slow isoforms of the thick-filament-associated protein, myosin binding protein-C. Extraction of myosin binding protein-C from skinned muscle normalized myofilament Ca²⁺ sensitivity of the KO extensor digitorum longus muscle. Thus, our data suggest that the M-band region of titin affects the expression of genes involved in the regulation of skeletal muscle contraction.     

Comprehensive analysis of titin protein isoform and alternative splicing in normal and mutant rats

Titin is a giant protein with multiple functions in cardiac and skeletal muscles. Rat cardiac titin undergoes developmental isoform transition from the neonatal 3.7 MDa N2BA isoform to primarily the adult 2.97 MDa N2B isoform. An autosomal dominant mutation dramatically altered this transformation. Titins from eight skeletal muscles: Tibialis Anterior (TA), Longissimus Dorsi (LD) and Gastrocnemius (GA), Extensor Digitorum Longus (ED), Soleus (SO), Psoas (PS), Extensor Oblique (EO), and Diaphram (DI) were characterized in wild type and in homozygous mutant (Hm) rats with a titin splicing defect. Results showed that the developmental reduction in titin size is eliminated in the mutant rat so that the titins in all investigated skeletal muscles remain large in the adult. The alternative splicing of titin mRNA was found repressed by this mutation, a result consistent with the large titin isoform in the mutant. The developmental pattern of titin mRNA alternative splicing differs between heart and skeletal muscles. The retention of intron 49 reveals a possible mechanism for the absence of the N2B unique region in the expressed titin protein of skeletal muscle.     

Blockade of lysophosphatidic acid receptors LPAR1/3 ameliorates lung fibrosis induced by irradiation

The activation of innate immune responses is critical to host defense against microbial infections, wherein nucleic acid-sensing pattern recognition receptors recognize DNA or RNA from viruses or bacteria and activate downstream signaling pathways. In a search for new DNA-sensing molecules that regulate innate immune responses, we identified RNA-binding motif protein 3 (RBM3), whose role has been implicated in the regulation of cell growth. In this study, we generated Rbm3-deficient (Rbm3^-/-) mice to study the role of RBM3 in immune responses and cell growth. Despite evidence for its interaction with immunogenic DNA in a cell, no overt phenotypic abnormalities were found in cells from Rbm3^-/- mice for the DNA-mediated induction of cytokine genes. Interestingly, however, Rbm3^-/- mouse embryonic fibroblasts (MEFs) showed poorer proliferation rates as compared to control MEFs. Further cell cycle analysis revealed that Rbm3^-/- MEFs have markedly increased number of G2-phase cells, suggesting a hitherto unknown role of RBM3 in the G2-phase control. Thus, these mutant mice and cells may provide new tools with which to study the mechanisms underlying the regulation of cell cycle and oncogenesis.     

Adaptive changes in structure of skeletal muscles from adult <Emphasis Type="Italic">Sod1</Emphasis> homozygous knockout mice

Cu/Zn superoxide dismutase (SOD1), which is localized cytoplasmically and in the mitochondrial intermembrane space, is an enzyme that is critically important for superoxide free-radical elimination. Compared with age-matched wild-type littermates (Sod1 ^+/+ ), SOD1 homozygous knockout (Sod1 ^-/- ) mice have smaller body masses, heart and skeletal muscle masses, and muscle cross-sectional areas. At the light-microscopic level, cross sections of skeletal muscles from Sod1 ^-/- mice show no gross structural abnormalities. Following the staining of muscles of Sod1 ^-/- mice for succinate dehydrogenase (SDH) enzymatic activity, a grouping of SDH-positive fibers has been observed. Immunostaining for neural cell adhesion marker in the gastrocnemius muscle of Sod1 ^-/- mice has revealed a small number of atrophic denervated muscle fibers. No denervated fibers are observed in extensor digitorum longus (EDL), tibialis anterior, or plantaris muscles. An increase in mRNA expression levels of myogenin and acetylcholine receptor alpha has been detected in muscles in Sod1 ^-/- mice, but no changes in MyoD expression occur. Compared with fast oxidative fibers in EDL muscles of Sod1 ^+/+ mice, those of Sod1 ^-/- mice show increased accumulations of sub-sarcolemmal mitochondria. We conclude that the lack of SOD1 in adult Sod1 ^-/- mice does not result in extensive denervation of skeletal muscle fibers, although the distribution of fiber types is modified, and that fast oxidative fibers develop alterations in the amount and spatial distribution of sub-sarcolemmal mitochondria. This study was supported by NIA grant PO1-AG20591, by the Nathan Shock Center Contractility Core (NIA grant P30-AG13283), and by a Nathan Shock Center Pilot Award (to T. Kostrominova).     

Passive tension in cardiac muscle: contribution of collagen, titin, microtubules, and intermediate filaments.

The passive tension-sarcomere length relation of rat cardiac muscle was investigated by studying passive (or not activated) single myocytes and trabeculae. The contribution of collagen, titin, microtubules, and intermediate filaments to tension and stiffness was investigated by measuring (1) the effects of KCl/KI extraction on both trabeculae and single myocytes, (2) the effect of trypsin digestion on single myocytes, and (3) the effect of colchicine on single myocytes. It was found that over the working range of sarcomeres in the heart (lengths approximately 1.9-2.2 microns), collagen and titin are the most important contributors to passive tension with titin dominating at the shorter end of the working range and collagen at longer lengths. Microtubules made a modest contribution to passive tension in some cells, but on average their contribution was not significant. Finally, intermediate filaments contributed about 10% to passive tension of trabeculae at sarcomere lengths from approximately 1.9 to 2.1 microns, and their contribution dropped to only a few percent at longer lengths. At physiological sarcomere lengths of the heart, cardiac titin developed much higher tensions (> 20-fold) than did skeletal muscle titin at comparable lengths. This might be related to the finding that cardiac titin has a molecular mass of 2.5 MDa, 0.3-0.5 MDa smaller than titin of mammalian skeletal muscle, which is predicted to result in a much shorter extensible titin segment in the I-band of cardiac muscle. Passive stress plotted versus the strain of the extensible titin segment showed that the stress-strain relationships are similar in cardiac and skeletal muscle. The difference in passive stress between cardiac and skeletal muscle at the sarcomere level predominantly resulted from much higher strains of the I-segment of cardiac titin at a given sarcomere length. By expressing a smaller titin isoform, without changing the properties of the molecule itself, cardiac muscle is able to develop significant levels of passive tension at physiological sarcomere lengths.     

Deletion of Kinin B2 Receptor Alters Muscle Metabolism and Exercise Performance

Metabolic syndrome is a cluster of metabolic risk factors such as obesity, diabetes and cardiovascular diseases. Mitochondria is the main site of ATP production and its dysfunction leads to decreased oxidative phosphorylation, resulting in lipid accumulation and insulin resistance. Our group has demonstrated that kinins can modulate glucose and lipid metabolism as well as skeletal muscle mass. By using B2 receptor knockout mice (B2R^-/-) we investigated whether kinin action affects weight gain and physical performance of the animals. Our results show that B2R^-/- mice are resistant to high fat diet-induced obesity, have higher glucose tolerance as well as increased mitochondrial mass. These features are accompanied by higher energy expenditure and a lower feed efficiency associated with an increase in the proportion of type I fibers and intermediary fibers characterized by higher mitochondrial content and increased expression of genes related to oxidative metabolism. Additionally, the increased percentage of oxidative skeletal muscle fibers and mitochondrial apparatus in B2R^-/- mice is coupled with a higher aerobic exercise performance. Taken together, our data give support to the involvement of kinins in skeletal muscle fiber type distribution and muscle metabolism, which ultimately protects against fat-induced obesity and improves aerobic exercise performance.     

Titin isoform switching is a major cardiac adaptive response in hibernating grizzly bears

The hibernation phenomenon captures biological as well as clinical interests to understand how organs adapt. Here we studied how hibernating grizzly bears (Ursus arctos horribilis) tolerate extremely low heart rates without developing cardiac chamber dilation. We evaluated cardiac filling function in unanesthetized grizzly bears by echocardiography during the active and hibernating period. Because both collagen and titin are involved in altering diastolic function, we investigated both in the myocardium of active and hibernating grizzly bears. Heart rates were reduced from 84 beats/min in active bears to 19 beats/min in hibernating bears. Diastolic volume, stroke volume, and left ventricular ejection fraction were not different. However, left ventricular muscle mass was significantly lower (300 +/- 12 compared with 402 +/- 14 g; P = 0.003) in the hibernating bears, and as a result the diastolic volume-to-left ventricular muscle mass ratio was significantly greater. Early ventricular filling deceleration times (106.4 +/- 14 compared with 143.2 +/- 20 ms; P = 0.002) were shorter during hibernation, suggesting increased ventricular stiffness. Restrictive pulmonary venous flow patterns supported this conclusion. Collagen type I and III comparisons did not reveal differences between the two groups of bears. In contrast, the expression of titin was altered by a significant upregulation of the stiffer N2B isoform at the expense of the more compliant N2BA isoform. The mean ratio of N2BA to N2B titin was 0.73 +/- 0.07 in the active bears and decreased to 0.42 +/- 0.03 (P = 0.006) in the hibernating bears. The upregulation of stiff N2B cardiac titin is a likely explanation for the increased ventricular stiffness that was revealed by echocardiography, and we propose that it plays a role in preventing chamber dilation in hibernating grizzly bears. Thus our work identified changes in the alternative splicing of cardiac titin as a major adaptive response in hibernating grizzly bears.     

The titin N2B and N2A regions: biomechanical and metabolic signaling hubs in cross-striated muscles

Muscle specific signaling has been shown to originate from myofilaments and their associated cellular structures, including the sarcomeres, costameres or the cardiac intercalated disc. Two signaling hubs that play important biomechanical roles for cardiac and/or skeletal muscle physiology are the N2B and N2A regions in the giant protein titin. Prominent proteins associated with these regions in titin are chaperones Hsp90 and αB-crystallin, members of the four-and-a-half LIM (FHL) and muscle ankyrin repeat protein (Ankrd) families, as well as thin filament-associated proteins, such as myopalladin. This review highlights biological roles and properties of the titin N2B and N2A regions in health and disease. Special emphasis is placed on functions of Ankrd and FHL proteins as mechanosensors that modulate muscle-specific signaling and muscle growth. This region of the sarcomere also emerged as a hotspot for the modulation of passive muscle mechanics through altered titin phosphorylation and splicing, as well as tethering mechanisms that link titin to the thin filament system.     

Method for isolation of intact titin (connectin) molecules from mammalian cardiac muscle

Cardiac titin was isolated from rabbit and ground squirrel ventricular muscles by a method that was used earlier to obtain myofibrils with intact minor proteins located in A-bands of sarcomeres (Podlubnaya, Z. A., et al. (1989) J. Mol. Biol., 210, 655–658). Small pieces of cardiac muscle were incubated for 2–3 weeks at 4°C in Ca²⁺-depleting solution before their homogenization to decrease activity of Ca²⁺-dependent proteases. Then the muscle was homogenized, and titin was isolated by the method of Soteriou, A., et al. (1993) J. Cell Sci., 14, 119–123. In control experiments, titin was isolated from cardiac muscle without its preincubation in Ca²⁺-depleting solution. Sometimes control titin preparations contained only T2-fragment, but generally they contained ～5–20% N2B-isoform of titin along with its T2-fragment. Preparations of titin obtained from rabbit cardiac muscle by our method contained ～30–50% of N2BA- and N2B-titin isoforms along with its T2-fragment. The content of α-structures in titin isolated by our method was increased. Actomyosin ATPase activity in vitro increased in the presence of titin preparations containing more intact molecules. This result confirms the significant role of titin in the regulation of actin-myosin interaction in muscles. The method used by us to preserve titin might be used for isolation of other proteins that are substrates of Ca²⁺-dependent proteases.     

Rbfox-regulated alternative splicing is critical for zebrafish cardiac and skeletal muscle functions

Rbfox RNA binding proteins are implicated as regulators of phylogenetically-conserved alternative splicing events important for muscle function. To investigate the function of rbfox genes, we used morpholino-mediated knockdown of muscle-expressed rbfox1l and rbfox2 in zebrafish embryos. Single and double morphant embryos exhibited changes in splicing of overlapping sets of bioinformatically-predicted rbfox target exons, many of which exhibit a muscle-enriched splicing pattern that is conserved in vertebrates. Thus, conservation of intronic Rbfox binding motifs is a good predictor of Rbfox-regulated alternative splicing. Morphology and development of single morphant embryos were strikingly normal; however, muscle development in double morphants was severely disrupted. Defects in cardiac muscle were marked by reduced heart rate and in skeletal muscle by complete paralysis. The predominance of wavy myofibers and abnormal thick and thin filaments in skeletal muscle revealed that myofibril assembly is defective and disorganized in double morphants. Ultra-structural analysis revealed that although sarcomeres with electron dense M- and Z-bands are present in muscle fibers of rbfox1l/rbox2 morphants, they are substantially reduced in number and alignment. Importantly, splicing changes and morphological defects were rescued by expression of morpholino-resistant rbfox cDNA. Additionally, a target-blocking MO complementary to a single UGCAUG motif adjacent to an rbfox target exon of fxr1 inhibited inclusion in a similar manner to rbfox knockdown, providing evidence that Rbfox regulates the splicing of target exons via direct binding to intronic regulatory motifs. We conclude that Rbfox proteins regulate an alternative splicing program essential for vertebrate heart and skeletal muscle functions.     

Comprehensive lipidomics in apoM-/- mice reveals an overall state of metabolic distress and attenuated hepatic lipid secretion into the circulation

Apolipoprotein M (apoM) participates in both high-density lipoprotein and cholesterol metabolism. Little is known about how apoM affects lipid composition of the liver and serum. In this study, we systemically investigated the effects of apoM on liver and plasma lipidomes and how apoM participates in lipid cycling, via apoM knockout in mice and the human SMMC-7721 cell line. We used integrated mass spectrometry-based lipidomics approaches to semiquantify more than 600 lipid species from various lipid classes, which include free fatty acids, glycerolipids, phospholipids, sphingolipids, glycosphingolipids, cholesterol, and cholesteryl esters (CEs), in apoM^-/- mouse. Hepatic accumulation of neutral lipids, including CEs, triacylglycerols, and diacylglycerols, was observed in apoM^-/- mice; while serum lipidomic analyses showed that, in contrast to the liver, the overall levels of CEs and saturated/monounsaturated fatty acids were markedly diminished. Furthermore, the level of ApoB-100 was dramatically increased in the liver, whereas significant reductions in both ApoB-100 and low-density lipoprotein (LDL) cholesterol were observed in the serum of apoM^-/- mice, which indicated attenuated hepatic LDL secretion into the circulation. Lipid profiles and proinflammatory cytokine levels indicated that apoM^-/- leads to hepatic steatosis and an overall state of metabolic distress. Taken together, these results revealed that apoM knockout leads to hepatic steatosis, impaired lipid secretion, and an overall state of metabolic distress.     

<Superscript>1</Superscript>H, <Superscript>15</Superscript>N and <Superscript>13</Superscript>C backbone and side chain resonance assignments of the RRM domain from human RBM24

Tissue development requires the expression of a regulated subset of genes, and it is becoming clear that the process of alternative splicing also plays an important role in the production of necessary tissue-specific isoforms. However, only a few of these tissue-specific splicing factors in mammals have so far been discovered. One of these factors is the RNA-binding protein RBM24 which has been recently identified as a major regulator of alternative splicing in cardiac and skeletal muscle development. The RBM24 protein contains an RNA recognition motif (RRM) domain that presumably mediates the binding to target pre-mRNA required for regulation of the splicing patterns. Here we report ¹H, ¹⁵N and ¹³C chemical shift assignments of the backbone and sidechain atoms for the RRM domain from human RBM24. Secondary chemical shift analysis and relaxation measurement confirm the canonical architecture of the RRM domain. The data will allow for atomic level studies aimed at understanding splicing regulation of target genes in heart and muscle development and investigation into a separate role of RBM24 in modulating mRNA stability of genes involved in the p53 tumor suppressor pathway.