Abstract

   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.

   Keywords: Titin, RBM20, Titin binding partners, Calcium handling,
   Intracellular calcium concentration

Introduction

   Pre-mRNA splicing is an important biological process that contributes
   to protein diversity in eukaryotic organisms [37]^1. This process is
   determined by RNA-binding splicing factors to produce protein isoforms
   in a tissue-specific and developmental-regulated manner [38]^2.
   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 [39]^3. 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 [40]^1, and RNA binding motif 20 (RBM20) is one of the
   muscle tissue specific splicing factors that has been identified
   recently [41]^4. Protein RBM20 contains two highly conserved functional
   domains, an RNA-recognition domain 1 (RRM1) and a serine (S)/arginine
   (R) rich domain (SR-domain) [42]^5. These structural features defined
   it as a serine/arginine (SR) protein that is essential for assembly in
   the spliceosome of pre-mRNA splicing [43]^6^, [44]^7. RBM20 is highly
   expressed in striated muscles, with the highest expression levels in
   cardiac muscle [45]^4. Rbm20 deficiency in animal models leads to
   dilated cardiomyopathy, a major cause of heart failure and a
   significant source of mortality and morbidity worldwide [46]^4^,
   [47]^8, and mutations in RBM20 have been identified in human patients
   with familial dilated cardiomyopathy [48]^4^, [49]^5^, [50]^9^,
   [51]^10^, [52]^11^, [53]^12^, [54]^13. Given the role of RBM20 in the
   regulation of gene splicing, efforts toward the identification of its
   RNA targets have been made [55]^4^, [56]^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 [57]^4^, [58]^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 [59]^15^-[60]^16^,
   [61]^17^-[62]^18. It has been increasingly recognized as a major
   disease-causing gene [63]^16^, [64]^19. Titin runs through distinct
   sarcomere regions: the Z-band, the I-band, the A-band and the M-band
   regions [65]^20^, [66]^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 [67]^22^, [68]^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”
   [69]^24. The ratio changes of these two isoforms are a major
   determinant for diastolic passive stiffness during left ventricular
   filling [70]^15^-[71]^18^, [72]^25. Aberrant ratio changes of titin
   isoforms have been found in both systolic and diastolic heart failure
   [73]^1^, [74]^26^-[75]^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.

Materials and methods

Animals and Sample preparation

   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 [76]^33.

Gel electrophoresis

   Titin isoforms were resolved using a previously published method using
   a vertical sodium dodecyl sulfate (SDS)-1% agarose gel electrophoresis
   (VAGE) system [77]^33. 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 [78]^33.

Microarray hybridization and data analysis

   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
   [79]^34. 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.
   [80]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 [81]^35.

Figure 2.

   [82]Figure 2
   [83]Open in a new tab

   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.

   The complete data set is publicly available in the NCBI Gene Expression
   Omnibus ([84]http://www.ncbi.nlm.nih.gov/geo/; accession numbers
   [85]GSE11137). Differential gene expression analysis was performed with
   Limma (R/Bioconductor) Gene set and pathway enrichment analysis was
   performed using DAVID [86]^36
   ([87]http://david.niaid.nih.gov./david/beta/index.htm) and GSEA
   ([88]http://software.broadinstitute.org/gsea/index.jsp)

Cardiomyocyte isolation and intracellular calcium measurements

   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 [89]^11. 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 Na[2]HPO[4], 1.2 MgSO[4], 12 NaHCO[3], 10 KHCO[3], 10 HEPES,
   and 30 taurine, pH 7.4) equilibrated with 5%CO[2]-95% O[2], 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 CaCl[2]) 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 CaCl[2]). 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 CaCl[2]), and Ca^2+ 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 Ca^2+ 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 Ca^2+ clearing rate. Single exponential curve fit
   programs were used to derive the intracellular Ca^2+ decay constant
   [90]^37.

Quantitative real time PCR (QPCR)

   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 [91]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 [92]^38.

Western Blot

   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 [93]^34.

Statistical analysis

   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.

Results and Discussion

RBM20 knockout Rat model characterization

   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 [94]^15^-[95]^18. Titin has two classes of
   isoforms with one smaller isoform N2B that is stiffer, and the larger
   isoforms N2BAs that are more compliant [96]^24^-[97]^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. [98]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. [99]1A and [100]1C). Percentage of N2B
   to total titin (N2B+N2BA) changes from about 2% at E18 to approximately
   85% at adult (Fig [101]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.
   [102]1B and [103]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
   [104]^20. 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 [105]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 [106]^39^-[107]^40.

Figure 1.

   [108]Figure 1
   [109]Open in a new tab

   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.

   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 [110]^4.
   Additionally, Ours and other studies have identified over thirty RBM20
   target genes that undergo alternative splicing [111]^4^, [112]^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.

Gene expression profile indicated that developmental changes of gene
expression are consistent with titin splicing pattern regulated by RBM20

   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. [113]1. The
   experimental design is shown in Fig [114]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
   [115]S2). Among 381 genes, 77 of them are differentially expressed at
   d1 (Supplement table [116]S3), 121 at d20 (Supplement table [117]S4),
   and 227 at d49 (Supplement table [118]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. [119]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. [120]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. [121]1A).

Gene functional analysis showed that up-regulated genes are associated with
heart failure and gene expression pattern is similar at later maturation
stages

   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 [122]^41. 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.
   [123]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. [124]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. [125]3E, 1A and 1B).

Figure 3.

   [126]Figure 3
   [127]Open in a new tab

   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.

Differentially expressed genes associated with titin direct and indirect
binding and these binding partners play diverse roles that may together
contribute to heart failure progression induced by RBM20

   Titin binds over 70 proteins directly and indirectly [128]^23. 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. [129]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
   [130]^42^-[131]^45. Gene MyBPC2 encodes a fast skeletal isoform
   (fMyBP-C) that is expressed primarily in fast skeletal muscles
   [132]^45. However, it can also be expressed in cardiac muscle, and its
   increased expression in the heart is associated with severe dilated
   cardiomyopathy [133]^46. Mammalian cardiac muscle contains two myosin
   isoforms: α-myosin and β-myosin encoded by two distinct genes [134]^47.
   In adult human cardiac muscle, the major isoform is β-myosin [135]^48.
   Elevated β-myosin level has been found in human failing heart
   myocardium, cardiomyopathy, aging and pressure overload
   [136]^49^-[137]^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 [138]^52, which has been found
   associated with titin N2B domain [139]^53 and the mitogen-activated
   protein kinase signaling cascade (Raf1/Mek1/2/Erk2) [140]^54. 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 [141]^54^-[142]^55.
   Carp and Darp are two members of the three homologous
   muscle-ankyrin-repeat proteins (MARPs) that interact with titin I-band
   N2A region [143]^56^-[144]^57. They are associated with muscle stress
   signaling and up-regulated when muscles undergo mechanical or metabolic
   challenge [145]^56. 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 [146]^32. 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 [147]^46^,[148]^49^, [149]^50^, [150]^55^,
   [151]^56, which was consistent with the phenotype of Rbm20 knockout
   rats and human patients with Rbm20 loss-of-function mutations [152]^4.

Figure 4.

   [153]Figure 4
   [154]Open in a new tab

   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.

   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 [155]^58^-[156]^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 [157]^62^-[158]^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 [159]^51^, [160]^64^-[161]^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.

Validation of titin binding partners and non-titin binding partners by QPCR
and western blotting

   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. [162]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. [163]5D). All five
   genes were significantly increased at d20 and d49 from both DNA
   microarray and QPCR (Fig. [164]5E and [165]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. [166]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.
   [167]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. [168]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.

Figure 5.

   [169]Figure 5
   [170]Open in a new tab

   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.

Figure 6.

   [171]Figure 6
   [172]Open in a new tab

   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.

RBM20 regulates a transcriptional network of calcium handling genes in the
heart and alters intracellular calcium levels during contraction

   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.
   [173]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. [174]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 [175]^4. 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. [176]6C), while, in response to
   electrical stimulus, intracellular calcium change was lower in Hm
   cardiomyocytes than that in Wt (Fig. [177]6D). However, intracellular
   calcium decay rate had no significant difference between Wt and Hm
   cardiomyocytes (Fig. [178]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 [179]^66^-[180]^68.
   However, the RyR channel is also positively controlled by the luminal
   side of the channel [181]^66^, [182]^69^-[183]^70. Auxiliary proteins
   (Trdn, Junctin and Casq) with luminal localization mediate the effect
   of luminal calcium on RyR [184]^71^-[185]^72. Casq is a major calcium
   binding protein and changes conformation upon calcium binding,
   resulting in interaction with RyR to sense luminal calcium
   [186]^73^-[187]^76. Trdn and Junctin mediate the inhibitory
   interactions between Casq and the RyR channel [188]^73. 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 [189]^14, 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 [190]^77. 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. [191]6E).

Conclusion

   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 [192]^78^-[193]^79, and trans- and cis-acting
   factors can regulate RNA polymerase II elongation [194]^80^, [195]^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.

Supplementary Material

   Supplementary tables.
   [196]Click here for additional data file.^ (271.8KB, pdf)

Acknowledgments