Functional analysis of a gene-edited mouse model to gain insights into the disease mechanisms of a titin missense variant

Abstract

Titin truncating variants are a well-established cause of cardiomyopathy; however, the role of titin missense variants is less well understood. Here we describe the generation of a mouse model to investigate the underlying disease mechanism of a previously reported titin A178D missense variant identified in a family with non-compaction and dilated cardiomyopathy. Heterozygous and homozygous mice carrying the titin A178D missense variant were characterised in vivo by echocardiography. Heterozygous mice had no detectable phenotype at any time point investigated (up to 1 year). By contrast, homozygous mice developed dilated cardiomyopathy from 3 months. Chronic adrenergic stimulation aggravated the phenotype. Targeted transcript profiling revealed induction of the foetal gene programme and hypertrophic signalling pathways in homozygous mice, and these were confirmed at the protein level. Unsupervised proteomics identified downregulation of telethonin and four-and-a-half LIM domain 2, as well as the upregulation of heat shock proteins and myeloid leukaemia factor 1. Loss of telethonin from the cardiac Z-disc was accompanied by proteasomal degradation; however, unfolded telethonin accumulated in the cytoplasm, leading to a proteo-toxic response in the mice.We show that the titin A178D missense variant is pathogenic in homozygous mice, resulting in cardiomyopathy. We also provide evidence of the disease mechanism: because the titin A178D variant abolishes binding of telethonin, this leads to its abnormal cytoplasmic accumulation. Subsequent degradation of telethonin by the proteasome results in proteasomal overload, and activation of a proteo-toxic response. The latter appears to be a driving factor for the cardiomyopathy observed in the mouse model.

Expression Signatures of Long Noncoding RNAs in Left Ventricular Noncompaction

“…HSPA8 (Hsc-70) is reported to be a titin interacting protein in an LVNC gene-edited mice model. Therefore, this gene could have a critical role in pathogenesis ( 32 ). Trophinin associated protein (TROAP) is shown to be involved in myocardial infarction ( 33 ), thus, its role in LVNC could also be important.…”

“…However, the roles of these pathways have not been extensively studied in relation to the development of LVNC ( 27 , 28 ). Furthermore, GSEA analysis identified HSPA1, DMD, and ADCY1 as having significant relevance in developing heart ailments and which presumably play a critical role in developing LVNC ( 32 , 34 , 36 ).…”

Phosphorylation at Serines 157 and 161 Is Necessary for Preserving Cardiac Expression Level and Functions of Sarcomeric Z-Disc Protein Telethonin

“…A potential post-translational mechanism for the reduced telethonin expression level in the hearts of HOM mice is a greater susceptibility of non-phosphorylated telethonin protein to degradation by intracellular pathways. Previous studies indicate that telethonin protein is subject to degradation by the UPS in multiple cell models ( Heng et al, 2008 ; Polge et al, 2018 ; Jiang et al, 2021 ). To explore the potential role of the UPS system in regulating telethonin protein expression level in the presence and absence of phosphorylation at S157/S161, we determined the effects of the UPS inhibitor MG132 ( Rock et al, 1994 ) in ventricular myocytes that were isolated from the hearts of WT and HOM mice and maintained in short-term culture.…”

“…Proteasome inhibition has also been shown to inhibit MDM2-mediated degradation of telethonin in H1299 cells ( Tian et al, 2006 ). Furthermore, a recent study has identified telethonin as a target of the proteasome in a titin missense variant mouse model of cardiomyopathy, where degradation of unbound, cytoplasmic telethonin by the proteasome results in proteasomal overload ( Jiang et al, 2021 ). In line with these observations, our study has identified UPS as an important post-translational mechanism responsible for the reduced telethonin expression level in the HOM mice; the proteasome inhibitor MG132 was found to increase significantly the expression level of S157/161A telethonin protein in myocytes from HOM mice, but not the expression level of phosphorylatable telethonin protein in myocytes from WT mice.…”

“…However, we did not observe any significant differences in the induction of pro-apoptotic genes (p53, p21) in response to chronic ISO treatment ( Knoll et al, 2011 ), or in calcium transient dynamics in isolated myocytes from WT and KI mice (data not shown) ( Ibrahim et al, 2013 ; Candasamy et al, 2014 ). It is possible that proteasomal overload due to increased degradation of mutant telethonin protein in our KI model may contribute to the observed cardiac phenotype ( Jiang et al, 2021 ), and further studies are warranted to delineate the principal mechanisms.…”