Reducing RBM20 activity improves diastolic dysfunction and cardiac atrophy
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Impaired diastolic filling is a main contributor to heart failure with preserved ejection fraction (HFpEF), a syndrome with increasing prevalence and no treatment. Both collagen and the giant sarcomeric protein titin determine diastolic function. Since titin’s elastic properties can be adjusted physiologically, we evaluated titin-based stiffness as a therapeutic target. We adjusted RBM20-dependent cardiac isoform expression in the titin N2B knockout mouse with increased ventricular stiffness. A ~50 % reduction of RBM20 activity does not only maintain cardiac filling in diastole but also ameliorates cardiac atrophy and thus improves cardiac function in the N2B-deficient heart. Reduced RBM20 activity partially normalized gene expression related to muscle development and fatty acid metabolism. The adaptation of cardiac growth was related to hypertrophy signaling via four-and-a-half lim-domain proteins (FHLs) that translate mechanical input into hypertrophy signals. We provide a novel link between cardiac isoform expression and trophic signaling via FHLs and suggest cardiac splicing as a therapeutic target in diastolic dysfunction.
Increasing the length of titin isoforms improves ventricular filling in heart disease.
FHL proteins are regulated via RBM20 and adapt cardiac growth.
RBM20 is a therapeutic target in diastolic dysfunction.
KeywordsHeart failure Therapy Mouse models RNA processing Hypertrophy signaling
Cardiovascular disease is the main cause of death worldwide with increasing prevalence of heart failure . Multiple environmental and genetic factors contribute to heart failure including age, sex, diabetes, kidney disease, inflammation, and mutations in sarcomeric proteins such as titin or cardiac splice factors such as the RNA binding motif 20 (RBM20) that regulates titin-based stiffness . The giant sarcomeric protein titin contributes to the diastolic properties of the heart. Titin undergoes extensive posttranslational modifications and alternative splicing adapts its elastic properties to the demands of the organism [3, 4]. The elastic PEVK and N2B regions support diastolic function, while differentially affecting cardiac growth [5, 6]. The PEVK region serves as an entropic spring, while the N2B region improves efficiency of the cardiac cycle via altered calcium sensitivity [7, 8]. Changes in titin isoform expression relate primarily to the elastic PEVK, N2B, and inter-adjacent immunoglobulin (IG) regions and are mediated by RBM20, the first splice factor related to human heart disease . Patients with mutations in Rbm20 express more compliant titin isoforms associated with dilated cardiomyopathy, fibrosis, and sudden cardiac death [9, 10]. Both, a naturally occurring RBM20-deficient rat strain and mice carrying a deletion of the RBM20 RNA recognition motif (RRM), express similar giant titin isoforms and recapitulate human RBM20 deficiency [2, 11].
In mice, increased diastolic compliance associated with longer titin isoforms contributes to improved cardiac function . In patients, the shift in titin isoform-expression from the stiff N2B to the more compliant N2BA isoform is also associated with improved function [4, 12]. This change in titin-based elasticity compensates for the increased ventricular stiffness by fibrosis. To evaluate if cardiac splicing could serve as a therapeutic target to decrease titin-based stiffness and could provide a lasting beneficial effect on diastolic dysfunction, we crossed the splice-deficient Rbm20∆RRM mouse and the titin N2B knockout mouse (Titin N2B−/−). Excision of the elastic N2B element of titin affects the mechanical properties of the sarcomere, hypertrophy signaling, and ultimately leads to a restrictive filling pattern . In double-deficient mice (Titin∆N2B/∆N2B Rbm20∆RRM/WT), reduced splicing with expression of more compliant titin isoforms had several positive effects. Not only was diastolic compliance improved but also cardiac dimensions, RNA levels of genes related to the cAMP response, and oxidative phosphorylation were restored. These findings suggest that RBM20 could be a therapeutic target in diastolic dysfunction.
Materials and methods
Mice were sacrificed by cervical dislocation at 100 to 120 days of age. The hearts were rapidly excised, washed in PBS, and dissected into atria, septum, right and left ventricle, and tissues were snap frozen in liquid nitrogen and stored at −80 °C. Mice were age and sex matched for each analysis (100–120 days old males). Generation of animal models and phenotyping by histology, echocardiograpy and conductance catheter is described in the supplement. All experiments involving animals were carried out following the Guide for the Care and Use of Laboratory Animals of the German animal welfare act and protocols were approved by the Committee on the Ethics of Animal Experiments of Berlin State authorities (LaGeSo).
Analysis and quantification of RNA and protein expression
Proteins and RNA were extracted from left ventricular tissue powder. Proteins were separated and blotted as described previously . Antibodies were used according to manufacturer’s instructions and are listed in Supplemental Table 1. Analysis of titin isoform expression was performed as described previously . TaqMan probes (Supplemental Table 2), qRT-PCR conditions and analysis, as well as RNAseq and global splicing analysis are described in the supplement.
Data are expressed as mean ± SEM. Multiple group comparisons were analyzed by two-way ANOVA. P values ≤ 0.05 were considered statistically significant.
All mouse RNA sequencing data have been submitted to the NCBI sequence read archive (SRP091317).
Reduced RBM20 expression restores cardiac dimensions in titin N2B-deficient mice
The heart-to-body-weight ratio of the N2B-KO is reduced , while heterozygous or homozygous deletion of the RBM20-RRM domain does not alter cardiac size . Introduction of the RBM20∆RRM allele into the N2B-KO reverts cardiac atrophy in splice-rescue animals: The heart-to-body-weight ratio is similar to wildtype levels (Fig. 1c) and ventricular geometry is restored as determined by trichrome staining of longitudinal cardiac slices (Fig. 1d). Neither the histology nor the real-time PCR for collagen isoform 1a2 (Supplemental Fig. S1c) provides evidence for ventricular fibrosis, which was reported in rats with a heterozygous deletion of RBM20 . Trophic changes in the heart are usually accompanied by the upregulation of hypertrophy markers such as atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP). In N2B-KO mice, ANP and BNP messenger RNA (mRNA) levels are similar to WT mice, but both are significantly elevated in left ventricles of RBM20-HET and even more in splice-rescue mice compared with WT (Supplemental Fig. S1c). Myofiber thickness was not significantly different between genotypes (Supplemental Fig. S1d).
Diastolic function is improved in splice-rescue mice
Splice-rescue mice exhibit molecular adaptations to improve energy metabolism
Decreased functional RBM20 protein shifts cardiac isoform expression
The main target of RBM20-dependent splicing in the mouse is titin. The heterozygous deletion of RBM20’s RRM leads to an up to 4-fold increase in PSI values of alternative exons in titin’s elastic region located between the N2B element and the PEVK region (Fig. 5d). Several transcripts are differentially spliced by RBM20, with exon skipping or alternative exon inclusion in Camk2d, Ldb3, Ttc17, Obscn, and Ank3 (Fig. 5e–i). Camk2d and Ldb3 have been described as RBM20 targets in the Rbm20-deficient rat [2, 14] and obscurin—although not significantly regulated in the rat—has been identified based on the physical interaction of the transcript with RBM20 . The newly identified RBM20-dependent isoforms expressed from the Ttc17, Ank3, Fhl1, Arhgap10, Cflar, Hmgb1, and Myh7 gene (Fig. 5; Supplemental Fig. S4 and S6) relate to actin polymerization, sarcomere structure and mechanotransduction. A summary of the gene functions and exons/domains affected is provided in Supplemental Table 5. Importantly, we find one differentially expressed isoform of the titin binding protein FHL1, with differential inclusion of the first exon that includes the ATG (Fig. 5j). RBM20 deficiency would increase inclusion of the exon containing the translation start and thus increase FHL1 protein expression.
FHL1 expression is increased in splice-rescue hearts
Diastolic dysfunction is an important contributor to the pathophysiology of heart failure, and our recent identification of RBM20 as a titin splice factor has provided a unique target to adjust the diastolic properties of the heart . We chose a genetic approach to adjust alternative splicing and concertedly affect multiple protein isoforms that adapt cardiac function in the titin N2B-KO mouse. In this animal model, the loss of titin’s heart-specific elastic N2B region leads to a complex cardiac phenotype including diastolic dysfunction . Combining the N2B mutant with a splice-directed therapeutic approach allowed us to address the following questions: first, can increased titin-based stiffness resulting from loss of one elastic domain be compensated by the inclusion of another structural region? Second, can synergistic adaptation of multiple mRNA isoforms restore a complex cardiac phenotype?
In the splice-rescue mouse, the diastolic dysfunction resulting from the loss of the titin N2B domain is completely restored, as determined by echocardiography and conductance catheter analysis. It includes the reduced E/A ratio as a sign of improved ventricular filling, normalized passive pressures, and normalized pressure volume relations. These findings not only suggest that adjusting exon inclusion via inhibition of RBM20 can improve the biomechanical properties of the heart. But also, they indicate that restoration of compliance does not have to occur at the protein-domain that caused the increase in stiffness, as the adjustment of PEVK and IG domain-based elasticity can compensate for the altered N2B-based stiffness. Furthermore, our data also indicate that even a reduction of RBM20 function to only 50 % is sufficient to restore diastolic function, increasing the therapeutic window of an RBM20-directed therapy. These findings are significant in light of the series of unsuccessful attempts to significantly improve cardiac function in patients with diastolic dysfunction [17, 18, 19, 20]. Finally, the reduced RBM20 activity affects the whole organism but does not lead to an obvious extracardiac phenotype (weight, fertility, grooming), indicating a sufficiently large therapeutic window for the future development of an RBM20 inhibitor.
In N2B-KO animals, reduced RBM20 activity not only improved diastolic dysfunction but also reverted the cardiac atrophy of titin N2B-deficient diastolic dysfunction model, illustrated by heart-to-body-weight measurements, trichrome stainings, echocardiography and hemodynamic catheter analysis. On the molecular level, we found a positive effect on metabolic function as reflected in reverted expression of genes related to fatty acid oxidation. This improvement could result from a direct effect of the differential splicing of RBM20 substrates that relate to cardiac hypertrophy signaling. As a secondary effect, improved diastolic function might change ventricular geometry as filling is improved or reduce strain of the ventricular wall that might lead to improved coronary flow and thus increased availability of nutrients. On a molecular level, altered titin-based elasticity could change localization or stability of proteins that differentially bind to the extended vs. relaxed titin filament .
We used RNAseq analysis to evaluate which transcripts are alternatively spliced dependent on RBM20. At the exon level, we find several genes affected, with more than 50 potential novel substrates of RBM20. These genes relate to the regulation of relaxation of cardiac muscle and myofibril assembly, which could explain part of the beneficial effect on the phenotype. Several genes are relevant to sarcomere structure and the hypertrophy response. In addition to candidate genes with relation to the improved cardiac phenotype, we thus identified several RBM20 substrates, including Obscurin, which was not significantly affected on the exon level in the RBM20-deficient rat  but has been identified by the CLIP analysis . For select transcripts, we found differential exon inclusion between RBM20-deficient and splice-rescue animals, which indicates a feedback of increased titin-based stiffness in the N2B-KO on alternative splicing and a potential primary role in the restored cardiac function of splice-rescue animals. Finally, the first exon level analysis of the titin N2B knockout revealed an unexpected finding with increased inclusion of I-band exons that would partially revert the increased stiffness resulting from the loss of the N2B exon. As RBM20 protein levels are not different between N2B knockout and wildtype animals, this effect could be mediated at the posttranslational level or via an RBM20-independent splice factor.
Secondary changes reflected in our analysis of the RNAseq data at the gene level can indicate adverse effects of targeting RBM20 or beneficial effects. Genes that are reverted, namely deregulated in N2B-KO but no longer in the splice-rescue animals, are indicative of a compensatory regulation. They relate to the regulation of fatty acid oxidation and the cellular response to cAMP. The reversion of genes related to beta oxidation affects all steps of the electron transfer chain, suggesting a concerted regulation resulting in improved energy balance in the splice-rescue animals. Thus, positive effects of a splice level therapeutic approach via RBM20 could synergistically improve diastolic function, size and metabolism.
To investigate the molecular basis of the trophic phenotype, we investigated the regulation of several hypertrophy pathways and their contribution to the restored cardiac size of the splice-rescue animals. We have previously proposed that the decreased trophic signaling in the N2B-KO heart is caused by decreased protein levels of the titin N2B-binding protein FHL2 . Interestingly, not only the deletion of the N2B region led to a decrease in FHL2 protein levels but also the heterozygous deletion of RBM20’s RRM—although the effects were not additive (splice-rescue mice expressed similar amounts of FHL2 as the N2B-KO). Thus, altered FHL2 protein levels are not sufficient to explain the increased cardiac trophic signaling in splice-rescue mice. In addition to the decreased FHL2 protein level, we found a minor increase of the titin binding protein FHL1 and a highly significant increase of its alternative splice isoform FHL1B in splice-rescue mice (~7.5-fold increase compared with WT). FHL1 mRNA levels were only mildly upregulated in splice-rescue mice and the FHL1 isoform that differentially includes the ATG-exon to improve translation only accounts for <10 % of the total FHL1 mRNA. Thus, the increase in FHL1 protein levels occurs at a posttranscriptional level and would be consistent with increased translation or decreased degradation of FHL1. FHL1 binds to titin’s N2B region, where it has been suggested to form a complex, which induces hypertrophy via Raf, MEK1/2 and ERK2 [15, 21]. Here, we do not find significant changes in ERK2 activity. Thus, the isolated increase in FHL1 protein levels in the absence of titin’s N2B region links FHL1 to mechanically induced trophic signaling that is independent on the N2B region as a force sensor and the induction of Erk phosphorylation. How the combination altered titin-based wall stress and deregulation of FHL1 can increase trophic signaling to restore cardiac size will have to be resolved in future experiments.
We thank B. Goldbrich-Hannig, C. Judis, J. Fröhlich, S. Schelenz, M. Taube, and A. Schiche for expert technical assistance, Arnd Heuser for support with cardiac phenotyping, and Claudia Langnick and Mirjam Feldkamp (Wei Chen lab, Max Delbrück Center for Molecular Medicine) for the sequencing runs. We are grateful to F. Luft for helpful discussions and for support in preparing the manuscript file. The Deutsche Forschungsgemeinschaft, Bonn, Germany (M.G.), the European Research Council grant StG282078 (M.G.), the Bundesministerium für Bildung und Forschung, Berlin, Germany (CaRNAtion, M.G.), and the NIH R01HL062881 (H.G.) supported this work.
Compliance with ethical standards
Conflict of interest
The authors have declared that no conflict of interest exists.
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