Abstract 13073: Antisense Transcripts Regulate Titin Alternative Splicing and Sarcomere Function in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Abstract

The giant protein Titin is an essential structural component of the sarcomere and a key determinant of passive tension during diastole. Titin tension is regulated by alternative splicing within the spring-like domains in the I-band region of TTN pre-mRNA. The cardiac RNA-binding protein RBM20 has been shown to promote TTN exon skipping during splicing, but additional factors are likely involved. We hypothesized that a natural antisense transcript in the TTN locus ( TTN-AS1 ) could regulate TTN splicing in cis through recruitment of spliceosome components. Through single nuclei RNA-Seq and RNA fluorescence in situ hybridization (ISH) on human cardiac tissue and iPS-derived cardiomyocytes (iPS-CM), we showed that the expression of TTN-AS1 was restricted to cardiomyocyte nuclei. The percent spliced in (PSI) of each TTN exon was calculated based on RNA-Seq data from iPS-CM (n=3) and 26 exons (all in the I-band region) that are normally spliced out were included to a significantly higher extent (adjusted p<0.05) upon siRNA-mediated TTN-AS1 knock down. Reduced exon skipping from TTN exon 50 was confirmed with exon-junction spanning qPCR probes (p<0.05, n=3). Similar effects on splicing were seen in cells where RBM20 had been knocked down. The consequences of TTN-AS1 knock down on cardiomyocyte contractile function was assessed using sarcomere tracking. Transgenic expression of ACTN2-GFP was used to visualize z-disks in iPS-CM, sarcomeres (n=14,636) were recorded during contractions using wide-field epifluorescence microscopy and analyzed using Sarc-Graph software. The analysis revealed that in cells where TTN-AS1 had been knocked down, sarcomere length and fractional shortening, as well as contraction and relaxation time were increased (all p<0.001). Again, similar effects were seen in iPS-CM where RBM20 had been knocked down. We observed co-localization of TTN-AS1 and RBM20 protein during active TTN splicing in iPS-CM nuclei using multiplex RNA ISH and immunofluorescence and confirmed interaction between TTN-AS1 and RBM20 using RNA Immunoprecipitation and qPCR. In conclusion, we show that TTN-AS1 can affect cardiomyocyte contractile properties and sarcomere dynamics through regulation of TTN alternative splicing.