Journal of Molecular and Cellular Cardiology 81 (2015) 107–113

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Review article

Insights into alternative splicing of sarcomeric genes in the heart Cornelis J. Weeland, Maarten M. van den Hoogenhof, Abdelaziz Beqqali, Esther E. Creemers ⁎ Experimental Cardiology, Academic Medical Center, Meibergdreef 15, 1105AZ Amsterdam, The Netherlands

a r t i c l e

i n f o

Article history: Received 4 December 2014 Received in revised form 15 January 2015 Accepted 5 February 2015 Available online 12 February 2015 Keywords: Sarcomere RBM20 RBM24 Alternative splicing Titin

a b s t r a c t Driven by rapidly evolving technologies in next-generation sequencing, alternative splicing has emerged as a crucial layer in gene expression, greatly expanding protein diversity and governing complex biological processes in the cardiomyocyte. At the core of cardiac contraction, the physical properties of the sarcomere are carefully orchestrated through alternative splicing to fit the varying demands on the heart. By the recent discovery of RBM20 and RBM24, two major heart and skeletal muscle-restricted splicing factors, it became evident that alternative splicing events in the heart occur in regulated networks rather than in isolated events. Analysis of knockout mice of these splice factors has shed light on the importance of these fundamental processes in the heart. In this review, we discuss recent advances in our understanding of the role and regulation of alternative splicing in the developing and diseased heart, specifically within the sarcomere. Through various examples (titin, myomesin, troponin T, tropomyosin and LDB3) we illustrate how alternative splicing regulates the functional properties of the sarcomere. Finally, we evaluate opportunities and obstacles to modulate alternative splicing in therapeutic approaches for cardiac disease. © 2015 Elsevier Ltd. All rights reserved.

Contents 1.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1. Titin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2. The RNA binding protein RBM20 orchestrates titin splicing . . . . . . . . 1.3. Myomesin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.4. Cardiac troponin T . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5. Tropomyosin . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.6. LDB3/Cypher/ZASP . . . . . . . . . . . . . . . . . . . . . . . . . . 1.7. RBM24 is a major regulator of alternative splicing in the developing heart . 1.8. Future directions . . . . . . . . . . . . . . . . . . . . . . . . . . . Disclosures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction The vast majority, over 90% of human genes expresses multiple mRNAs via alternative splicing, a post-transcriptional mechanism to generate multiple proteins from individual genes [1]. It is estimated that approximately 100,000 different proteins are produced from only 21,000 protein-coding genes [2]. The importance of alternative splicing ⁎ Corresponding author at: Experimental Cardiology, Room L2-108-3, Academic Medical Center (AMC), Meibergdreef 15, 1105AZ Amsterdam, The Netherlands. Tel.: +31 20 5668544. E-mail address: [email protected] (E.E. Creemers).

http://dx.doi.org/10.1016/j.yjmcc.2015.02.008 0022-2828/© 2015 Elsevier Ltd. All rights reserved.

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is exemplified by the fact that the human genome harbors fewer genes than the rice genome, which suggests that organism complexity depends greatly on post-transcriptional diversification of gene products [3]. During constitutive splicing, non-coding sequences called introns are cleaved from the pre-mRNA transcript and the coding sequences (i.e. exons) that remain are merged to form a mature mRNA. The splicing process is carried out by a ribonucleoprotein complex called the spliceosome. This complex of 5 small nuclear ribonucleic particles (snRNPs) together with other proteins assemble on a pre-mRNA transcript at splice sites to cleave the introns and fuse the remaining exons. During alternative splicing different combinations of exons may

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be included or excluded from the final mRNA. Consequently, the proteins translated from these alternatively spliced mRNAs contain different amino acid sequences resulting in proteins with different stability, intracellular localization or even distinct functions [1]. Each alternative splicing event is carried out by the spliceosome, but influenced by a combination of sequence-specific RNA binding proteins (RBPs), which have the ability to bind directly to the pre-mRNA. The splicing ‘choices’ are further regulated by cis-acting regulators, i.e. the exonic and intronic sequences that flank the spliced elements and can either promote or repress splicing by influencing spliceosome recruitment to a splice site. The function of these sequences is often context dependent, meaning that a sequence can promote or repress splicing depending on its location relative to the spliced element. Interestingly, numerous RBPs that bind these splicing sequences are expressed in a cell type-specific manner, allowing the generation of cell type-specific mRNA isoforms [4]. Alternative splicing is not a rigid process, and many proteins can switch isoforms by the balanced action of RBPs to meet the everchanging demands of the cell. Kong et al. [5] were the first to demonstrate that alternative splicing is broadly altered in human heart failure. By interrogating mRNA splicing using exon arrays, they found a significant decrease in the efficiency of mRNA splicing in heart failure. Splicing of key sarcomere genes, which encode for proteins comprising the basic contractile unit of muscle, such as troponin T, β-myosin heavy chain, filamin C and titin was found to be altered, and it has been suggested that this plays a causal role in heart failure pathology [5]. In 2009, the first mutation was identified in a cardiac splicing factor (i.e. RNA binding motif protein 20, RBM20), which was associated with human heart failure, and the underlying mechanism involved altered splicing of the giant sarcomeric protein titin [6,7]. Also during cardiac development, alternative splicing programs are used to adapt to changing physiological conditions [8,9]. For example, the passive stiffness of mammalian hearts increases during the course of development, an effect that is achieved by adjusting the expression of stiff titin splice variants [10–12]. Intriguingly, hypertrophic or failing hearts often revert to the expression of fetal splice variants. In this regard, isoform switches in sarcomeric proteins are an important feature of the ‘fetal gene program’ that the heart activates upon stress [13]. Recently, another muscle-specific splicing factor was identified: RNA binding motif protein 24 (RBM24), which was shown to govern numerous muscle-specific splicing events [14]. The critical involvement of these splicing events in cardiac development was revealed in RBM24 knockout mice, which fail to form sarcomeres in cardiomyocytes and die around embryonic day 13.5 due to cardiac malformations. Our knowledge of alternative splicing and its role in the heart has grown rapidly the last decade. Especially advances in next-generation sequencing contributed to this, since this technology enables investigating

Z-line

Troponin T

1.1. Titin Titin, with its ~ 3000 kDa weight and over 1 μm in length, is the largest protein in the human body [15]. Many of its 363 exons undergo alternative splicing, allowing the assembly of various possible titin proteins [16]. It forms a molecular spring that spans half of the sarcomere of striated muscle between the M-line and Z-line (Fig. 1) [17]. Its size and elasticity make titin an important player in maintaining structural integrity of the sarcomere. Moreover, it delivers most of the passive tension that restores the sarcomere to normal length after extension and it provides elastic recoil after contraction [18–20]. Interestingly, cardiac titin undergoes alternative splicing primarily within its elastic regions, resulting in two main classes of titin: the short and stiff N2B isoform, and the longer, more compliant N2BA isoforms [10,15]. Isoforms can be co-expressed, allowing a full spectrum of variability in passive stiffness [21]. In mammalian fetal hearts, the long N2BA isoform, also called fetal cardiac titin is predominantly expressed (see Table 1) [10]. After birth, fetal N2BA is gradually replaced with stiffer N2B, which is associated with increased myocardial passive stiffness [10–12]. Increased passive stiffness is needed after birth to prevent the growing filling pressures (preload) from overfilling the ventricles during diastole [11]. Thus, increasing the relative expression of stiffer titin isoforms provides a way to adapt cardiac function after birth. Work of the group of Granzier indicated that this titin switch also enhances contractility. Sarcomere length-dependent myofibrillar Ca2+-sensitivity, the basis of the Frank– Starling mechanism, is higher when short isoforms are expressed [22]. The isoform switch therefore seems to influence both systolic and diastolic cardiac function [11,22]. Remarkably, the diseased adult heart undergoes a reverse titin isoform transition. In this regard, in transplanted hearts of coronary artery disease patients, and in patients with dilated cardiomyopathy (DCM) and heart failure, the fraction of the fetal titin isoform N2BA is substantially higher than in controls [23–25]. Is this also an adaptive response to disease to improve cardiac function? A recent study that investigated cardiac function in mice with artificially increased N2BA expression suggests that it is [26]. It was found that under conditions of exercise,

M-line

Actin

Titin

alternative splicing at the genome-wide level and the interrogation of protein-RNA interactions. In this review, recent advances in understanding alternative splicing of sarcomeric genes in cardiac development and disease will be discussed. Through various examples (titin, myomesin, troponin T, tropomyosin and LDB3, schematically illustrated in Fig. 1), we illustrate how alternative splicing impacts functional properties of the sarcomere. We also speculate about future developments in this field and we discuss how the regulation of alternative splicing may develop as a potential therapeutic target in heart disease.

Tropomyosin

Myosin

Myomesin

Fig. 1. The cardiac sarcomere, illustrating the structural and contractile proteins discussed.

Z-line

LDB3/Cypher

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109

Table 1 Sarcomeric alternative splicing events that occur during development and disease and their functional consequences. Protein

Splice events

(Speculated) functional consequence

Fetal heart

Adult heart

Diseased heart

Titin

N2B:N2BA ratio low

N2B:N2BA ratio high

N2B:N2BA ratio ↓ in DCM & coronary artery disease

Myomesin

EH-myomesin high

EH-myomesin low

EH-myomesin ↑ in DCM

Cardiac troponin T

cTnT1 high cTnT1 → cTnT3 cTnT2 low cTnT4 low TPM1κ possibly high

cTnT3 high Other isoforms not expressed

cTnT4 ↑ in HF

TPM1α high and TPM1κ low in striated muscle

TPM1κ ↑ in DCM

Cypher2c low Cypher2c ↑ (short isoform) in mice

Cypher2c high

Splicing events that occur in HF are not known.

Tropomyosin

LDB3/Cypher

increasing titin compliance improves diastolic function and overall cardiac function, despite an impaired systolic function [26]. 1.2. The RNA binding protein RBM20 orchestrates titin splicing The observation that aberrant titin splicing is associated with DCM and the fact that alternative splicing of titin is a key determinant of myocardial passive stiffness stimulated the search for RNA binding proteins that determine the titin isoform transitions. In 2012, Guo and colleagues identified such a titin splice factor in a rat strain with a spontaneously occurring mutation that associated with longer titin isoforms and the development of DCM [7]. Using genetic mapping they revealed that these rats have a 95-kb deletion removing exons 2–14 of the striated muscle-enriched gene encoding RBM20. RNA sequencing in these rats revealed that RBM20 not only regulates alternative splicing of titin's spring region, but a variety of cardiac mRNAs, including CaMKIIδ, CACNA1C and the sarcomeric LDB3 [7]. It is likely that missplicing of these RNAs also affects their cellular functions. Subsequent studies gained mechanistic insights into splicing regulation by RBM20 [27,28]. For instance, it was found that RBM20 acts as a splicing repressor by binding to a consensus sequence (UCUU) found near 3′and 5′ splice sites within introns [28]. Also in humans, mutations in RBM20 have been associated with DCM [6,29,30]. In fact, RBM20 mutations have been detected in 3% of human subjects with DCM. Like the rat mutants, patients carrying an RBM20 mutation also showed increased expression of the longer titin isoforms [7]. It has been suggested that upregulating compliant isoforms of titin may serve as an attractive therapeutic approach for lowering diastolic stiffness and improve diastolic filling in patients with heart failure with preserved ejection fraction (HFpEF) [31]. This might be accomplished through the inhibition of RBM20 activity, for example by using small-molecules that interfere with RBM20 binding to titin pre-mRNA. Nevertheless, caution should be applied since titin isoform switching will also impact other important properties of the heart such as a reduction in titin-dependent diastolic recoil and decreased contractility through attenuation of the Frank–Starling mechanism [26,32]. Whether modulation of titin isoform expression accomplished through inhibition of RBM20 could serve as a potential therapy to treat patients with HFpEF is an interesting question to explore in future studies. Another RBM20 target, calcium/calmodulin regulated kinase IIδ (CaMKIIδ), is a key enzyme involved in calcium handling, gene transcription, and signaling processes in the heart. Eleven isoforms have been identified, of which

When N2B:N2BA ratio high: – passive stiffness increased – length-dependent Ca2+-sensitivity increased When N2B:N2BA ratio low: – systolic function decreased – diastolic function increased EH-myomesin low: – contractile efficiency increased – M-line stability decreased cTnT3 ↑ → Ca2+-sensitivity decreased cTnT4 ↑ in HF: – co-expression of cTnT isoforms- contractile efficiency decreased TPM1κ ↑: – fractional shortening of LV decreased – calcium sensitivity decreased – actin-binding affinity decreased – systolic and diastolic dysfunction Functional role of Cypher2c unknown. Less PKC binding, less PGM1 in short isoforms

four (δA, δB, δC, and δ9) are present in the heart [33]. CaMKIIδA is primarily localized in the T-tubules where it can, for example, mediate the Ca2+-dependent positive feedback loop (also known as ‘facilitation’) of the L-type calcium channel (LTCC) [34]. CaMKIIδB localizes primarily to the nucleus where it can phosphorylate transcription factors and impact gene expression [35]. CaMKIIδC localizes primarily to the cytosol where it can phosphorylate proteins of the sarcoplasmatic reticulum (SR) and the sarcomere [36,37]. It must be noted, however, that this is far from complete, and that the different isoforms also overlap in function and localization [38]. To date, there is very little known about the function and localization of CaMKIIδ. Overall, it is evident that regulation of CaMKIIδ splicing, for example by RBM20 or ASF/SF2 can have profound effects on the heart by affecting Ca2+ handling, gene transcription and other processes [7,9]. 1.3. Myomesin The sarcomeric M-line contains myomesin proteins which are known to crosslink myosin filaments to the M-line to maintain structural integrity during contraction [39]. Myomesin also interacts with titin and mediates signaling in response to stretch [39,40]. The myomesin family comprises three genes: MYOM1, MYOM2 and MYOM3, of which MYOM1 undergoes alternative splicing by inclusion of an EH segment of about 100 amino acids in its center [41,42]. The EH domain of myomesin acts as a molecular spring, similar to the PEVK domain in titin, through which myomesin elasticity can be increased [43]. EHmyomesin is the main myomesin isoform in embryonic hearts and in slow skeletal muscle. However, cardiac EH-myomesin expression is downregulated around birth and myomesin isoforms lacking the EH domain are upregulated after birth (see Table 1) [41]. Strikingly, the timing of the downregulation of EH-myomesin coincides with the fetal titin isoform switch [41]. It is proposed that expression of shorter myomesin, together with shorter titin, improves the longitudinal alignment of the contractile filaments, and increases contraction efficiency of the sarcomere [39,44]. However, why longer isoforms are preferred in fetal hearts is not fully understood. Agarkova & Perriard have performed extensive research on myomesin and posed an interesting hypothesis that EH-myomesin expression benefits the sarcomere by creating a heterogeneous or ‘fuzzy’ appearance that broadens its working range and increases sarcomere stability [39,45]. Here, fuzzy refers to the disordered alignment of contractile elements resulting from variable actin length and more elastic M-band bridges that gives the M-band a fuzzy appearance on EM images [45]. This fuzzy M-band lowers contractile

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efficiency, but increases sarcomere stability under stretch. It is argued that increased stability is needed in fetal hearts as well, because the fetal myofibrils are prone to overstretching. For a detailed overview of the studies that support this hypothesis we refer to the excellent review by Agarkova and Perriard [39]. Re-expression of fetal EH-myomesin occurs in DCM and heart failure, which coincides with the switch in titin isoforms during DCM, suggesting the involvement of a shared adaptive program [46]. In this regard, myomesin was recently identified as another direct target of RBM20 [28]. Upregulation of EH-myomesin in DCM is associated with a higher degree of M-line elasticity and may contribute to progressive ventricular dilation. Whether EH-myomesin upregulation is a cause or consequence of DCM remains unknown. Following the earlier mentioned ‘fuzzy sarcomere’ hypothesis, it could be the latter. The dilated heart must contract in a stretched state. The EH-myomesin upregulation therefore could be an adaptive response to increase sarcomere stability and make the heart better equipped to work under stretch. On the other hand, upregulation of EH-myomesin in mouse models for DCM occurs early, suggesting it may have a causal role [46]. Perhaps the answer lies somewhere in between, where EH-myomesin upregulation is intended as an adaptation to cardiac overstraining, but becomes maladaptive and actually contributes to disease progression. However, to test this hypothesis an EH-myomesin overexpression model is needed.

1.5. Tropomyosin Tropomyosins (TPMs) are a protein family encoded by 4 different genes (TPM1, TPM2, TPM3 and TPM4) that produce multiple protein products through alternative splicing. Tropomyosins are actin-binding proteins essential for calcium-dependent thin filament function and muscle contraction. In healthy individuals, the TPM1 gene undergoes tissue-specific alternative splicing, producing smooth muscle specific TPM1β and striated muscle specific TPM1α and TPM1κ. TPM1κ and TPM1α are structurally resembling, differing only by exon 2 [58]. The two isoforms are co-expressed in the heart, but TPM1κ is upregulated in patients with cardiomyopathy and heart failure [59]. In transgenic mice overexpressing TPM1κ, decreased fractional shortening of the left ventricle, decreased myofilament calcium sensitivity, weakened actin-binding affinity and resulted in systolic and diastolic dysfunction. This suggests a possible mechanism through which alternative splicing of tropomyosin may contribute to the development and progression of human DCM and heart failure [59]. A similar mechanism as described for troponin may be at play: an imbalance of different isoforms resulting in dysregulated calcium sensitivity, which in turn leads to desynchronized contraction [56]. TPM1 has recently been identified as a target of RBM20, but it is still unknown which exact TPM splice event is under the control of RBM20 [7]. An interesting next step would be to explore if RBM20 governs the expression ratios of the TPM1α and TPM1κ isoforms and if changes in its expression could modulate this ratio.

1.4. Cardiac troponin T 1.6. LDB3/Cypher/ZASP Troponin T is the tropomyosin-binding subunit of the troponin complex and plays a key role in the actin-myosin interactions responsible for muscle contraction [47]. Cardiac troponin T (cTnT) undergoes an isoform switch during perinatal development in mammals [48,49]. Alternative splicing of exons 4 and 5 gives rise to four human cardiac isoforms: cTnT1 (contains exons 4 and 5), cTnT2 (contains only exon 5), cTnT3 (contains only exon 4) and cTnT4 (misses both exons) [48,50]. The fetal heart predominantly expresses cTnT1 and to a lesser extent cTnT2 and cTnT4 [48,50]. During perinatal development, a regulated switch occurs from embryonic cTnT1 to adult cTnT3 through exon 5 exclusion, and cTnT3 becomes the only expressed isoform in the adult heart. Interestingly, the cTnT4 isoform expressed in the fetal heart becomes re-expressed in failing human hearts [48–51]. The different isoforms of cTnT are functionally distinct; exons 4 and 5, for instance, give rise to an N-terminal of cTnT that is more negatively charged, which results in higher Ca2 +-sensitivity compared to adult cTnT3 and cTnT4 [50]. This implies that cardiac troponin undergoes functional adaptations through isoform switches to fit altering cardiac demands in the context of development and disease. Such adaptations may be vital, as high Ca2 +-sensitivity is associated with cardiac arrhythmias [52]. The developmental transition of cTnT isoforms is regulated by the CELF family of RNA binding proteins, Muscleblind-like (MBNL1) and polypyrimidine-tract binding protein (PTB). These splice factors compete over exon 5 inclusion, in which CELF proteins promote and MBNL1 and PTB repress exon 5 inclusion. The balance between these competing factors determines alternative splicing [53–55]. Gradual downregulation of CELF proteins after birth results in increased exon 5 exclusion and thus relative cTnT3 increase [53]. It has been shown that co-existence of multiple cTnT variants with different Ca2+-sensitivity causes desynchronized activation of ventricular muscle and impairs cardiac efficiency proportional to the degree of cTnT heterogeneity, resulting in lower maximum left ventricular pressure, slower contractile and relaxation velocities, and decreased stroke volume [56,57]. This indicates that expression of a single troponin isoform, like in the normal adult heart, is a criterion for synchronized contraction and rhythmic pumping of the heart [56]. Therefore, the co-expression of isoforms that arise from cTnT4 upregulation could contribute to the development of heart failure.

The Z-line protein LIM domain binding 3 (LDB3), also called Cypher in mice and Z-line alternatively spliced PDZ-motif protein (ZASP) in humans, is yet another example of a gene that undergoes developmentally regulated alternative splicing [60]. It plays a role in muscle cell structure and function as it maintains structural integrity of the Z-line under the mechanical stress of contraction [61]. Three cardiac splice isoforms of Cypher (mouse LDB3) are known in humans and mice, the 2 long (Cypher1c and 3c) isoforms and 1 short isoform(2c). During mouse cardiogenesis the short isoform Cypher2c is barely present, but it is upregulated postnatally. The expression levels of the long isoforms are consistent throughout development [60]. The functional distinctions between the long and short isoforms are unknown. In mice, genetic deletion of long Cypher isoforms leads to late-onset DCM, and postnatal lethality is seen in about a third of the long Cypher knockout mice [62]. In contrast, deletion of the short Cypher2c isoform has no apparent effect. However, since the full Cypher knockout phenotype is more severe than the phenotype of only long Cypher knockouts, short Cypher 2c does appear to have functional significance [62,63]. Since the short Cypher2c lacks LIM domains that are important for PKC-binding, at least some functional difference between long and short isoforms can be explained by different PKC binding affinity [60,64]. Also, the short isoform Cypher2c includes exon 10, which the long isoforms Cypher 1c and Cypher 3c do not, and has been implicated in phosphoglucomutase 1 (PGM1) binding. PGM1 is a key enzyme in glucose consumption and energy management [65]. Further research is needed to explain the postnatal increase of short Cypher and discover individual functions of Cypher2c. Like titin, LDB3 splicing is regulated by RBM20 [7,28]. Inclusion of exon 4 (cardiac specific) or exons 5 and 6 (skeletal muscle specific), which are mutually exclusive, occurs through regulation by RBM20. Therefore, RBM20 determines the tissue-specific splicing of LDB3 rather than the ‘long versus short’ splicing events described above. Lack of RBM20 results in exclusion of exon 4 and expression of exons 5 and 6 [7]. The cardiac isoform (containing exon 4) is involved in the recruitment of PGM1 to the sarcomere under stress conditions. Mutations in exon 4 and exon 10 of LDB3 were identified in DCM patients and were associated with reduced PGM1 binding [64,65]. Similarly, the absence of exon 4 by RBM20-deficiency in mice may have contributed to the

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observed DCM phenotype [7,65]. Additionally, in the RBM20 knockout mice described by Methawasin et al., the short Cypher isoform was downregulated [26]. How specific isoforms of Cypher contribute to the Z-line integrity remains unknown [62]. 1.7. RBM24 is a major regulator of alternative splicing in the developing heart In an elegant study by the laboratory of T. Braun [14] it was recently shown that the splicing factor RBM24 governs a large number of muscle-specific splicing events. Targeted inactivation of RBM24 in mice disrupted cardiac development and resulted in embryonic lethality around embryonic day 13.5. These embryos revealed multiple cardiac malformations, such as ventricular-septum defects and reduced trabeculation and compaction of the ventricular walls. However, the most striking observation was the nearly complete loss of sarcomeres in mutant cardiomyocytes. This is in line with loss-of-function studies in zebrafish, which revealed that RBM24 deficiency resulted in a reduction of sarcomeric proteins, Z-disc abnormalities and diminished cardiac contractility [66]. RNA sequencing of hearts of RBM24 knockout mice (before onset of the phenotype) and in vitro splicing assays subsequently revealed that RBM24 governs at least 68 splicing events, mostly by promoting muscle-specific exon inclusion. Several of the spliced transcripts that rely on RBM24 have been reported to play crucial roles in cardiogenesis, sarcomere formation, hypertrophic or dilated cardiomyopathies or skeletal myopathies. Examples of RBM24 splicing targets that have been associated with heart biology include Naca, Fxr1, Abcc9, Slc25a3, Usp25 and Usp28 [67–72]. Embryonic lethality in the RBM24 KO mice precluded the analysis of RBM24 function in the adult heart, and also the functional analysis in disease models. Therefore, to shed light on the splicing events regulated by RBM24 in the adult heart, the generation and analysis of the conditional KO allele for RBM24 is awaited with great interest. 1.8. Future directions It has become evident that splicing events occur in regulated networks on a global scale, rather than in isolated events. For instance, the developmental stiffening of titin coincides with the stiffening of myomesin in the M-line and the splice events that underlie this stiffening seem both to be regulated by the splicing factor RBM20. Since titin and myomesin (but also other RBM20 targets such as LDB3) support sarcomere structure, this suggests the existence of a common structural ‘splice program’. Similarly, targets of RBM24 also share functionality and seem to govern alternative splicing of regulators of sarcomere formation rather than splicing of the structural sarcomeric proteins themselves [14]. It must be noted though that not all identified targets of RBM24 are necessarily direct targets, as their splicing could have been altered secondary to disease processes. Besides its function as a major regulator of alternative splicing, RBM24 has also been implicated in other posttranscriptional processes. In specific, it was recently shown that RBM24 enhances mRNA stability by direct binding to the 3′UTR of the p21 mRNA [73]. Whether the stability of other mRNAs is also regulated by RBM24 and whether the underlying mechanism involves the inhibition of microRNA accessibility to the 3′UTR remains to be seen. Alternative splicing is a wide-reaching regulatory system, which demands a broad approach in studying it. Due to recent advances in next-generation sequencing, we are now able to investigate transcriptome-wide splicing events in great detail. To identify the position of protein-RNA interaction on the (pre-)mRNA with high resolution, the CLIP assay (cross-linking and immunoprecipitation) has been developed. By coupling CLIP to high-throughput sequencing (CLIP-seq), the full landscape of interaction of any protein with RNA can be obtained. CLIP-seq has been used to identify numerous direct splicing targets of RBM20, and this technique also pinpointed the precise RBM20-RNA recognition element responsible for the RBM20-

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mediated splicing [28]. Several useful variants to this CLIP technique have been developed and have been reviewed elsewhere [74]. It is expected that the application of this assay to other splicing factors will further shed light on the broad splicing schemes that play a role in the developing and diseased heart. It is not known yet how many RNA binding proteins are involved in splicing control in any given cell, but considering the observation that up to 1000 different proteins can bind to messenger RNA in HeLa cells, we expect that there are many more (alternative) splicing factors to be uncovered [75]. The high degree of regulation makes alternative splicing a potential target for therapeutic use in heart disease. Of the methods used to interfere with splicing, the use of antisense oligonucleotides (AONs) receives much attention. AONs are designed in such a way that they bind to a specific splicing sequence to manipulate splicing. Duchenne's muscular dystrophy (DMD), caused by mutations in the dystrophin gene is the first disease in which AONs have been clinically tested. The administration of AONs to DMD patients promotes exon skipping of the mutated exon (to avoid premature truncation of the protein) and this even showed modest improvements in exercise capacity [76]. Other diseases in which correction of splicing has been accomplished through AONs in animal models include Progeria (LMNA) [77], Spinal Muscular Atrophy (SMN2) [78] and Myotonic Dystrophy (DMPK) [79]. As discussed earlier, upregulation of compliant isoforms of titin may also serve as an attractive therapeutic approach to improve diastolic filling in patients with heart failure with preserved ejection fraction. Since titin appears to be the most sensitive target of RBM20, it will be interesting to find out whether this can be accomplished by AONs, designed against the RNA recognition elements of RMB20 within titin mRNA [7]. Inhibiting RBM20 activity itself may also be a therapeutic option, as this will also increase the expression of titin-N2BA. Inquiring the optimal degree of RBM20 inhibition to achieve the desired effect on cardiac function would be interesting for future studies. A potential drawback of this approach may be the fact that RBM20 inhibition is too broad since it also affects splicing of numerous other important cardiac genes, such as CAMK2D [7]. Overexpressing specific splice isoforms of cardiac proteins (for example by the use of adeno-associated viruses) could also serve a therapeutic function. This will be impossible for titinN2BA due to its tremendous size, but there are other genes that generate specific splice isoforms that exert cardioprotective effects, such as calcineurin A (CnA) and insulin-like growth factor (IGF) [80,81]. In patients with RBM20 mutations, this same technique could be used to introduce normal RBM20 in the heart to restore alternative splicing back to normal. In contrast, downregulation of cTnT4, which is upregulated during heart failure, could be used to create a more uniform expression pattern of troponin T. This could benefit cardiac function, as heterogeneous expression of troponin T desynchronizes contraction and impairs contractile efficiency [50,56]. In conclusion, alternative splicing has emerged as an important layer of gene regulation. The recent identification of RBM20 and RBM24 as major regulators of muscle-specific splicing events has shed light on the importance of these fundamental processes in the heart. Further insights in our understanding as to how splicing of sarcomeric genes is regulated may eventually enable us to shift splicing towards increased expression of the cardioprotective isoforms to reduce pathological effects. Disclosures None. Acknowledgments This work was supported by grants from the Netherlands Organisation for Scientific Research (NWO) [grant numbers 825.13.007 and 836.12.002] and the Netherlands Cardiovascular Research Initiative [grant number CVON 2011-11].

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