Poster Presentation Inaugural Australian Ubiquitin Summit 2025

Investigation of TRIM55-associated hypertrophic cardiomyopathy in hiPSC-CMs (#122)

Antonia TL Zech 1 2 3 , Ellen B Keen 1 2 , Rachel Morris 1 2 , Pooja Venkat 1 2 , Giorgia Zappacosta 1 2 , James W McNamara 1 2 4 5 , Benjamin L Parker 4 5 , Keshava K Datta 6 , Petros Syrris 7 8 , Luis R Lopes 7 8 , Perry M Elliott 7 8 , Enzo R Porrello 1 2 4 5 , David A Elliott 1 2 3 9
  1. MCRI, Melbourne/Parkville, VIC, Australia
  2. Novo Nordisk Foundation Centre for Stem Cell Medicine (reNEW), Murdoch Children's Research Institute, Melbourne, Vic, Australia
  3. School of Biomedical Sciences and Department of Paediatrics, University of Melbourne, Melbourne, Vic, Australia
  4. Department of Anatomy and Physiology, University of Melbourne, Melbourne, Vic, Australia
  5. Centre for Muscle Research, University of Melbourne, Melbourne, Vic, Australia
  6. Bio21 Molecular Science and Biotechnology Institute, Melbourne, Vic, Australia
  7. Centre for Heart Muscle Disease, Institute of Cardiovascular Science, University College London, London, England, United Kingdom
  8. Barts Heart Centre, St.Bartholomew’s Hospital, Barts Health NHS Trust, London, England, United Kingdom
  9. Australian Regenerative Medicine Institute, Monash University, Melbourne, Vic, Australia

Variants in TRIM55, encoding a myogenic ubiquitin ligase, have been associated with hypertrophic cardiomyopathy (HCM), but the underlying disease mechanism is still unknown, limiting the identification of novel therapeutic interventions.

Aims: This study aims to dissect the molecular function of TRIM55 in cardiomyocytes and to understand how TRIM55 contributes to HCM pathogenesis.

Methods: TRIM55 Knockout (KO) and TRIM55 reporter human induced pluripotent stem cell (hiPSC) lines were generated and differentiated into cardiomyocytes. Full proteome, TRIM55 interactome and cardiac ubiquitinome mass spectrometry analysis, as well as bulk RNA sequencing was performed. Calcium handling analysis, live cell imaging and immunofluorescence imaging was carried out in cardiac monolayer, and the functional phenotype was assessed in 3D cardiac organoids.

Results: TRIM55-KO hiPSC-CMs displayed an HCM-like functional phenotype with abnormal calcium handling and, in 3D cardiac organoid, a prolonged relaxation time in comparison to healthy controls. OMICs profiling revealed dysregulation of both structural sarcomeric components and sarcomere regulators in TRIM55-KO hiPSC-CMs, suggesting a regulatory role of TRIM55 in sarcomere formation. Live cell and immunofluorescence imaging showed TRIM55 has a variegated localisation either at the M-band of the sarcomere or more diffuse punctate cytoplasmic localisation. Investigation of the TRIM55 interactome, confirmed the close interaction with the two close family members, TRIM54 and TRIM63. Further, the TRIM55-dependant ubiquitinome contains 515 proteins, including sarcomeric proteins found at the M-band. These data suggest the HCM phenotype is due to impaired sarcomere protein homeostasis.   

Conclusion: Our findings indicate that TRIM55 is vital for cardiac sarcomere maintenance and function.