RBM20, a gene for hereditary cardiomyopathy, regulates titin splicing

Abstract

Alternative splicing has a major role in cardiac adaptive responses, as exemplified by the isoform switch of the sarcomeric protein titin, which adjusts ventricular filling. By positional cloning using a previously characterized rat strain with altered titin mRNA splicing, we identified a loss-of-function mutation in the gene encoding RNA binding motif protein 20 (Rbm20) as the underlying cause of pathological titin isoform expression. The phenotype of Rbm20-deficient rats resembled the pathology seen in individuals with dilated cardiomyopathy caused by RBM20 mutations. Deep sequencing of the human and rat cardiac transcriptome revealed an RBM20-dependent regulation of alternative splicing. In addition to titin (TTN), we identified a set of 30 genes with conserved splicing regulation between humans and rats. This network is enriched for genes that have previously been linked to cardiomyopathy, ion homeostasis and sarcomere biology. Our studies emphasize the key role of post-transcriptional regulation in cardiac function and provide mechanistic insights into the pathogenesis of human heart failure.

Figure 1

Mapping of the titin splice defect and validation of RBM20 as the affected gene. (a) In a splice deficient rat strain the QTL resides in a 2.1 Mbps interval on the long arm of chromosome 1 (1q55). The coding sequence of all known genes included in the interval was unchanged except for the Rbm20 gene with a 95 kb deletion that eliminates all exons following exon 1. (b) Southern-blot of genomic DNA confirms the deletion with the loss of a 3 kb internal HindIII fragment in homozygous (−/−) and a reduced signal in heterozygous (+/−) compared to wildtype (+/+) rats. (c) Rbm20 RNA levels normalized to 18S and wildtype levels reflect the changes documented by Southern blot with a 76% reduction in the heterozygote and no expression in the homozygote (< 1%). N = 9. P < 0.01 (**) and P < 0.001 (***). (d) With reduced levels of Rbm20, there is a shift from the shorter N2B-titin to the larger N2BA isoforms (heterozygotes) and N2BA-G (homozygote mutant) as determined by agarose gel electrophoresis. T2 refers to a proteolytic fragment that is independent of the titin isoform. Gapdh was used as a loading control for the western blot. (e) Adenoviral gene transfer into homozygous deficient cardiomyocytes leads to reexpression of RBM20 in homozygous deficient neonatal cardiomyocytes (last lane). (f) Treatment of neonatal RBM20 deficient cardiomyocytes with Rbm20-vs. control-virus (C) resulted in a reproducible downregulation of the larger N2BA isoform starting from 24h after infection and upregulation of the N2B isoform (asterisks). (g) Quantification of titin isoform expression shows a linear increase in the N2B/N2BA-G ratio from 24 to 72h after infection with the Rbm20 adenovirus. One-Way-Anova test P < 0.001 (***).

Figure 2

Analysis of RBM20 expression and function. (a) In nuclei of HL1 cells Rbm20 partially colocalizes with the splice factor U2AF65 and Ptbp1, but not with additional splice factors tested (Cugbp1 and SC-35) or coilin, a marker for Cajal bodies. (Scale bar 5 μm). (b) Autoradiogram of 5′-32P-labeled RNA-protein immunoprecipitates (IPs) derived from RBM20 and IGF2BP1 overexpressing HEK293 cells. After cultivation in media containing a photoreactive nucleoside and UV-crosslinking both the positive control IGF2BP1 and RBM20 bound RNA. Untransfected HEK293 cells were used as a negative control (CTRL). (c) Specific activity of RBM20 on titin RNA was monitored in a cell-based splice reporter assay utilizing the 5′-PEVK exons, firefly and renilla luciferase (Fluc and Rluc). Expression of RBM20 leads to exclusion of the Fluc containing exon. Black lines indicate exon junctions as determined by sequencing RT-PCR products derived from cells that express the reporter construct with and without RBM20. (d) The ratio of Fluc to Rluc reflects splice activity and decreased with expression of RBM20 in both HEK293 cells and in C2C12 myoblasts. (e) The control RNA binding proteins and splice factors PTBP1 and HuD did not interfere with alternative splicing. (f) Transfection of increasing amounts of RBM20 (1x, 5x, or 25x excess of the expression construct) led to improved excision of the Fluc reporter. (g) Rbm20 protein expression at 2 months of age is largely restricted to cardiac and skeletal muscle (H, Heart, Q, Quadriceps, Ut Uterus, Thy Thymus, Lu Lung, Liv Liver, Kid Kidney, Spl Spleen, Br Brain). Actin shows proper regulation in muscle vs. non-muscle tissues and was used as a loading control. (h) C2C12 cells were stained for α-actinin (red) as a marker of sarcomere maturation and staged for low, medium, and high differentiation based on the fluorescence pattern. Rbm20 expression (green) was restricted to the nuclei (DAPI, blue). (Scale bar 5 μm). (i) Quantification of Rbm20 expression at four levels of cellular differentiation as shown in (h). The ratio of total intensity distribution over the nuclear area was lowest in undifferentiated cells (−), intermediate with both low and high differentiation (low, high), and highest at intermediate differentiation (med). Significance levels at P = 0.05 (*) and P < 0.001 (***) are indicated (N > 15 per group).

Figure 3

Signs of cardiomyopathy with arrhythmia and sudden death in Rbm20 deficient rats. (a, b) Left ventricular diameter in diastole (LVDd) as determined by echocardiography was increased in both heterozygous and homozygous mutants as a sign of dilated cardiomyopathy (P < 0.05; n = 15). Changes in LV diameter in systole (LVDs) and fractional shortening (FS), a parameter of contractile function, did not reach statistical significance. (c) Subendocardial fibrosis was present in heterozygote mutants (+/−) as indicated by the trichrome staining (blue). The fibrotic area was increased and compacted in the homozygous hearts (−/−). Size bar = 100 μm. (d) Interstitial fibrosis was significantly increased in LV from heterozygous (13% fibrotic area) and homozygous (26%) as compared to wildtype hearts (3%). N = 13. (e, f) Increased inducibility of arrhythmia in both hetero- and homozygotes was associated with an increase in sudden death starting from 10 months of age. Deaths in percent are indicated at 18 months of age. Log-rank (Mantel-Cox) test P = 0.03; n = 130.

Figure 4

(a) Identification of a novel RBM20 mutation in a human with severe cardiomyopathy and arrhythmia that leads to a change of serine to alanine within the RS domain. (b) The region affected is highly conserved between species. (c, d) The newly identified S635A mutation renders RBM20 inactive in our splice reporter assay – along with all previously described RBM20 mutations in exon 9. V535I resides in exon 6 and does not affect reporter activity. N=8; SEM. P-rich, proline rich; ZnF, Zinc Finger; RRM RNA-recognition-motif; RS, Arginine-Serine rich. (e) RBM20 deficiency leads to changes in titin isoform expression as determined by SDS-agarose gel electrophoresis. Compared to control subjects with cardiomyopathy of unrelated cause (CP1 and CP2), the RBM20 deficient individual (S635A) expresses a larger cardiac titin isoform. A similar increase is seen in the heterozygous mutant rat, while homozygote rats express the larger N2BA isoforms.

Figure 5

Characterization of RBM20 dependent isoform expression. (a) A conserved set of 31 genes with RBM20 dependent alternative splicing was identified by next-generation sequencing of humans and rats, which includes titin as the most differentially spliced gene. An enrichment analysis of these candidate substrates using gene ontology and medical subject heading terms (MeSH) suggests a role of RBM20 in regulating protein isoforms within the sarcomere and sarcoplasmic reticulum of striated muscle and a relation to heart failure and cardiomyopathy. (b) Alternative splicing of genes with high ΔPSI values as determined by RNA-seq was confirmed by RT-PCR. The shift between isoforms predominant in the wildtype (i1) and those primarily expressed in the mutant (i2) was strongest in homozygote Rbm20 deficient rats (−/−). Heterozygotes showed an intermediate effect (+/−). Rbm20 exon 1 (5′) and 6 to 7 (3′) were amplified as controls with only 3′ exons deleted in the mutant. PCR fragments were sequence verified as indicated in the Supplement Figure 9. (c) Relative exon expression was determined by qRT-PCR. Exons analyzed for Titin, CamkIIδ, Trdn, Camk2γ, Sorbs1, Sh3kbp1 were included in transcripts of homozyogtes (−/−) at higher levels. The alternatively spliced exon tested for Ldb3 was increasingly included in the wildtype (elevated expression in +/+). The strong effects of titin and CamkIIδ were confirmed by larger differences in exon inclusion and significance between all groups. Significance levels at P <= 0.05 (*) and P <= 0.001 (***) are indicated (n = 3 per group). Primers used are indicated in the supplementary figure S8.

Figure 6

Alignment of orthologous rat and human exons for Titin, CAMKIIδ, LDB3, and CACNA1C to compare RBM20 dependent isoform expression. PSI scores of humans are indicated in red for the RBM20 deficient individual (S635A) and in blue for two control subjects with DCM of unrelated cause (CP1 and CP2). The average PSI of 3 rats per genotype is provided below and aligned to the human exon structure (wildtype blue; heterozygote light red, homozygote dark red). The resulting ΔPSI for human (continuous line) and rat (dashed lines; light red wildtype vs. heterozygotes, dark red wildtype vs. homozygotes) show highly conserved deflections up (additional inclusion of exons) and down (exon skipping) in differentially spliced regions across species with heterozygotes at intermediate levels. (a) Titin is predominantly spliced in the elastic region extending from the N2B to the PEVK region. The 5′ and 3′ regions are unaffected as indicated by ΔPSI curves approaching 0. Both alternatively spliced and constitutive regions are conserved between species. (b) CAMKIIδ exons 14–16 are mutually exclusive and differentially regulated by RBM20 with exon14 up-regulated in the control and exons 15 and 16 in the mutant (indicated by the crossing of PSI lines between exon 14 and 15). (c) LDB3 undergoes a similar exon switch that affects exon 4 (included in the control) and exons 5 and 6 excluded with reduced RBM20 activity. (d) CACNA1C is consistently regulated in human and rat for the majority of alternatively spliced exons (8, 9*, 22, 31) but differences in inclusion rates are small with ΔPSI of <10.