Reduced Expression of MTSS1 Increases Sarcomere Number and Improves Contractility in Select Forms of Monogenic DCM

Background: The I-bar protein MTSS1 is a known modifier of heart failure and contractile phenotypes but its role in modulating contractile dysfunction in genetic forms of Mendelian dilated cardiomyopathy (DCM) is not known. Methods: The potential role of cardiac MTSS1 in TTN DCM was explored using time-to-event models in observational human datasets. Using induced siRNA and mutant forms of pluripotent stem cell cardiomyocytes (iPSC-CMs) the impact of siRNA knockdown of MTSS upon sarcomere and Cardiomyocyte biology was assessed via quantitative high-content microscopy, and the impact and mechanism of MTSS1 knockdown upon contractility was assessed using engineered heart tissues (EHTs). Results: Amongst individuals affected with TTN DCM, a variant conferring lower cardiac levels of MTSS1 was associated with significantly improved event-free survival from cardiovascular death or heart transplant (HR 0.29, p=0.0016). Knockdown of MTSS1 by siRNA significantly improved the appearance of iPSC-CM models of TTN (p=2.9e-06), CSRP3 (p=3.1e-14), and RBM20 (p=4.4e-04) DCM as assessed by quantitative microscopy. Correspondingly, siRNA knockdown of MTSS1 increased contractility in EHT models of TTN DCM (p=0.003), CSRP3 DCM (p=0.008), and RBM20 DCM (p<2e-16). Across all genetic backgrounds, knockdown of MTSS1 was observed to increase the number of sarcomeres (p<0.0001), and in co-immunoprecipitation experiments MTSS1 physically interacts with MYO18A a key determinant of early sarcomere formation. Knockdown of MTSS1 resulted in increased transcription of MYH7 (0.29 log2FC, p=2.9e-06) along with other sarcomere genes. Conclusions: In iPSC-CMs Knockdown of MTSS1 by siRNA increased number of sarcomeres and was observed to increase twitch force in select in vitro models, suggesting MTSS1 may have a previously unrecognized role in modulating sarcomere production or turnover. Human observational and iPSC-CM experimental data supports the hypothesis that reduced expression of MTSS1 may be beneficial in Mendelian DCM caused by TTN, RBM20, and CSRP3.