Heterozygous distal A-band TTN truncating variants cause greater doxorubicin-induced apoptosis, calpain activity, and contractility decline in hiPSC-CMs than I-band variants or controls.
Does doxorubicin induce greater apoptosis, calpain activity, and contractility impairment in hiPSC-CMs with A-band TTNtv compared to I-band TTNtv or controls?
In an hiPSC-CM model, truncating variants in the A-band of titin predispose to more severe doxorubicin-induced cardiotoxicity compared to I-band variants or controls.
Absolute Event Rate: 0% vs 0%
Abstract Background In dilated cardiomyopathy, truncating variants in titin (TTNtv) show position-dependent effects: proximal I-band (TTNtvI) harbour less pathogenic potential than distal A-band TTNtv (TTNtvA). TTNtv are present in 7-10% of patients with anthracycline-induced cancer therapy-related cardiac dysfunction (CTRCD). Purpose To assess if a location-dependent effect of TTNtv is at play in anthracycline-induced CTRCD, in order to refine risk stratification in patients. Methods Distinct isogenic TTNtv human induced pluripotent stem cell (hiPSC) lines were created using CRISPR/Cas9 starting from an in-house reprogrammed hiPSC line of a healthy female. Exon 48 (E48), located in the I-band, and exon 357 (E357), located in the A-band were targeted, based on TTNtv identified in our CTRCD population. The engineered TTNtvI and TTNtvA lines were validated. Differentiation of each iPSCs line to cardiomyocytes was performed at least two times. Expression of cardiac markers was evaluated with immunocytochemistry. For assessment of contractility, bright-field videos of contracting monolayers were obtained at day 27-44 (VOX microscope) and analysed using Musclemotion software. Next, doxorubicin (DOX) was added for 24 hours (0, 0. 1, 1 or 10 µM) and contractility imaging was repeated. After DOX-treatment, apoptosis (Caspase 3 activity) and calpain activity were assessed using luciferase assays (Caspase-Glo 3/7 Assay, Calpain-GloTM Protease Assay), as DOX induces calpain-dependent titin proteolysis. Results We created isogenic hiPSC lines with a heterozygous TTNtv located in the I-band: E48C1: c. 13836₁3853del and E48C2: c. 13836₁3838delTTTinsGATACATACAA, or with a heterozygous TTNtv located in the A-band: E357C1: c. 100373delT and E357C2: c. 100372₁00373insT. In all cell lines, DOX resulted in increased nuclear staining for distal titin, with a dose-response effect observed. Overall, cells became less elongated. Apoptosis significantly increased with increasing DOX dose and according to cell line, with lowest values in the isogenic control and highest in TTNtvA (Fig 1). Similarly, Calpain activity increased with higher DOX dose, and this was most outspoken in TTNtvA, present in TTNtvI, but absent in the control line. For contractility, DOX-dose and targeted exons showed a significant interaction (p0. 0001) indicating a different sensibility to DOX according to which exon was targeted. However, for control and TTNtvI lines, no linear DOX dose related effects were seen, while TTNtvA hiPSC-CMs showed worst contractility when treated with the highest. For each DOX dose, TTNtvA and TTNtvI hiPSC-CMs had significantly lower contractility than the control cell line (Fig 2). Conclusion DOX influenced cell architecture in all cell lines. Heterozygous TTNtvI and TTNtvA hiPSC-CMs show increased apoptosis and calpain activity in response to DOX treatment compared to an isogenic control cell line. Effects were more prominent in TTNtvA than in TTNtvI hiPSC-CMs. Figure 1 Figure 2
Boen et al. (Sat,) reported a other. Heterozygous distal A-band TTN truncating variants cause greater doxorubicin-induced apoptosis, calpain activity, and contractility decline in hiPSC-CMs than I-band variants or controls.