Speaker Dr Ay Lin Kho, Research Associate, Gautel Lab, Randall Centre for Cell and Molecular Biophysics

Title In vitro and in vivo studies to decipher the role of titin variants in cardiac and skeletal muscle disease

Host Elisabetta Brunello 

 

Abstract The giant protein titin is the largest protein in humans and acts as a molecular ruler and spring within the muscle sarcomere. Variants in the titin gene (TTN) are responsible for a range of skeletal and cardiac diseases, called titinopathies. Depending on their mode of inheritance and location in the highly spliced TTN gene, these range from severe congenital cardiomyopathies, skeletal myopathies or combinations of both, to the late or adult onset dilated cardiomyopathy (DCM) and milder skeletal myopathies.

Heterozygous TTN truncating variants (TTNtvs) are the single largest genetic cause of DCM (15-20% of all cases) and are involved in many congenital titinopathies. However, the clinical utility of TTNtvs is limited as the penetrance of these variants is low, with TTNtvs present in the general population at a frequency much higher than the incidence of DCM. Futhermore, the pathology of these truncating variants and whether they are expressed or subjected to nonsense mediated decay resulting in haploinsufficency is still unclear. Missense variants in TTN are responsible for several skeletal myopathies, and many congenital titinopathies show a compound heterozygous pattern of inheritance, with missense variants commonly inherited with a second titin variant in trans.

To tease apart the contribution of missense and truncating titin variants to disease, we investigated the effect of these mutations using biophysical and cellular methods as well as in vivo, studying patient biopsies and generating mice containing patient derived TTN variants. The mice heterozygous for the p.Asp32441Phefs*1 truncating variant are overtly healthy but this variant is embryonic lethal in homozygosity. qRT-PCR suggests the presence of mutant RNA but so far, we have no evidence for the presence of truncated titin within the sarcomere. Disease-linked missense variants destabilise their domain and result in aggregated protein when expressed in cells, and mice expressing the Trp34072Arg missense variant in homozygosity show upregulation of proteins involved in proteostasis. Both cardiac and skeletal muscle are particularly sensitive to proteostatic insult as they are non-dividing long-lived cells; we therefore propose a mechanism for titinopathies where one truncating variant in TTN causes an imbalance in the cell’s proteostasis status, and a further insult caused by a second (missense) TTN variant further tips the cell into collapsing proteostasis.