Final Report Summary - CARDIOSPLICE (A systems and targeted approach to alternative splicing in the developing and diseased heart: Translating basic cell biology to improved cardiac function) Cardiac function adapts to developmental, environmental, and pathophysiological changes. Alternative splicing plays a major role in this process exemplified by the perinatal isoform switch of titin, a protein critical for ventricular filling. We have used a naturally occurring rat strain deficient in titin splicing with persistent expression of the larger titin isoforms to identify RBM20 as the first splice factor that regulates titin isoform expression.We found that RBM20 is part of the major spliceosome where it interacts with a specific RNA motive and multiple RNA binding proteins and excludes select cardiac exons from transcripts relevant to sarcomere structure, contractile function, and signal transduction. Within the nucleus RBM20 partially co-localizes with the splice factors PTBP1 and U2AF6. Expression correlates with sarcomere assembly in differentiating myoblasts and peaks when α-actinin is largely localized in mature Z-bodies within the nascent myofiber (punctuate α-actinin staining) and declines as sarcomeres continue to mature - suggesting a role for RBM20 in regulating sarcomere assembly. RBM20 is predominant in striated muscle with the highest levels in the heart, where expression increases perinatally but is reduced in the adult. The maximum expression of RBM20 coincides with the switch in titin isoforms from fetal to adult.The RBM20 deficient rat recapitulates human cardiomyopathy with ventricular enlargement, arrhythmia, an increased rate of sudden death, and extensive fibrosis, attributable to impaired splicing of specific RBM20 substrates. This combined pathology has been described in a distinctive form of autosomal-dominant dilated cardiomyopathy (DCM), which maps to the syntenic locus on human chromosome 10q25. The underlying RBM20 mutations cluster within the RS domain of the protein, which is critical for splice activity. We found the P638L mutation in this region not only to eliminate splice activity but also to reduce protein levels to 50%. Similar Pro-to-Leu mutations have been shown to increase susceptibility to protein degradation.Our titin based splice reporter assay allows the functional characterization of RBM20 mutations and thus targeted recommendations for personalized therapy of patients with RBM20 mutations (up to 3% of patients with idiopathic DCM). Towards an improved therapy of these patients and patients with impaired filling of the heart in diastole, we evaluated RBM20 as a therapeutic target. In the N2B knockout, reducing RBM20 levels to 50% using a genetic approach improved diastolic function and restored heart size. Similar downregulation of RBM20 in a knockdown approach in vivo altered titin isoform expression. In a pharmacological approach, we utilized our splice-reporter assay to identify compounds that inhibit RBM20 activity and thus increase the size of titin in tissue culture. We are currently developing these molecules and extend our small molecule screen to derive optimized compounds for application in vivo.In summary, we have established a role for RBM20 in cardiac development and provide novel mechanistic insight into the pathogenesis of human heart failure and options for therapy.
