The first modifications of tubulin were discovered more than 40 year ago and consist in the catalytic removal and re-incorporation of the last C-terminal amino acid residue (tyrosine) of -tubulin. While the tubulin-tyrosine ligase (TTL) is known for 20 years, the identity of the tubulin carboxypeptidase complex (TCC) was only discovered recently (Aillaud C et al., Science 2017; Nieuwenhuis et al., Science 2017). TCC is composed of vasohibin (VASH1 or VASH2) and the small vasohibin-binding protein (SVBP), which is a chaperone required for vasohibin stability and function. Recent evidence indicates that the cycle of -tubulin tyrosination/detyrosination is critical for cell and tissue integrity, and defects have been implicated in cancer, brain disorganisation and heart failure. Specifically, it has been shown higher detyrosinated tubulin levels in failing human cardiac samples of various types (Robison P et al., Science 2016) and in human and mouse samples with hypertrophic cardiomyopathy (Schuldt M et al., Circ Heart Fail 2021). We also recently found VASH1 as the major isoform in the heart (Chen CY et al., Circ Res 2020), suggesting that targeting VASH1 or increasing TTL could be a therapeutic option for different forms of heart failire. To decipher the role of tubulin tyrosination/detyrosination in cardiomyocytes, we generated with CRISPR/Cas9 tools two distinct human induced pluripotent stem cell (iPSC) lines, one deficient in TTL and the other deficient in SVBP that is part of the tubulin-carboxypeptidase complex. Knock-out of TTL or SVBP was confirmed in iPSC-derived cardiomyocytes on mRNA and protein levels. Sarcomere structure and microtubules network were further analyzed by immunofluorescence, showing well-structured sarcomeres and different levels of tubulin detyrosination according to the genotype (TTL-KO > wild-type > SVBP-KO). These cell lines were further used to generate engineered heart tissues (EHTs). I will present several published and unpublished data, including gene therapy in mice.