Tubulin tyrosine ligase variant perturbs microtubule tyrosination, causing hypertrophy in patient-specific and CRISPR gene-edited iPSC-cardiomyocytes
Pratul Kumar Jain, Susobhan Mahanty, Harshil Chittora, Veronique Henriot, Carsten Janke, Minhajuddin Sirajuddin, Perundurai S. Dhandapany
Pratul Kumar Jain, Susobhan Mahanty, Harshil Chittora, Veronique Henriot, Carsten Janke, Minhajuddin Sirajuddin, Perundurai S. Dhandapany
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Research Article Cardiology Genetics Stem cells

Tubulin tyrosine ligase variant perturbs microtubule tyrosination, causing hypertrophy in patient-specific and CRISPR gene-edited iPSC-cardiomyocytes

Abstract

Hypertrophic cardiomyopathy (HCM) is a hereditary heart condition characterized by either preserved or reduced ejection fraction without any underlying secondary causes. The primary cause of HCM is sarcomeric gene mutations, which account for only 40%–50% of the total cases. Here, we identified a pathogenic missense variant in tubulin tyrosine ligase (TTL p.G219S) in a patient with HCM. We used clinical, genetics, computational, and protein biochemistry approaches, as well as patient-specific and CRISPR gene-edited induced pluripotent stem cell–derived cardiomyocytes (iPSC-CMs), to demonstrate that the TTL pathogenic variant results in a reduced enzymatic activity and the accumulation of detyrosinated tubulin leading to the disruption of redox signaling, ultimately leading to HCM. Our findings highlight — for the first time to our knowledge — the crucial roles of the TTL variant in cardiac remodeling resulting in disease.

Authors

Pratul Kumar Jain, Susobhan Mahanty, Harshil Chittora, Veronique Henriot, Carsten Janke, Minhajuddin Sirajuddin, Perundurai S. Dhandapany

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Figure 7

PTL treatment in TTL p.G129S iPSC-CMs normalizes α-tubulin detyrosination.

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PTL treatment in TTL p.G129S iPSC-CMs normalizes α-tubulin detyrosinatio...
(A) Immunofluorescence images of detyrosinated α-tubulin (cyan), α-sarcomeric actinin (green), phalloidin (red), and Hoechst (blue) were captured in WT, untreated, and treated TTL p.G219S iPSCs-CMs with PTL (10 mM for 2 hours). The WT, regardless of treatment, showed no significant differences. WT serves as a comparative control. Lower panel is the grayscale of the detyrosinated α-tubulin of the same image of the upper panel. Scale bar: 20 μm. (B) Immunofluorescence images of desmin (magenta), α-sarcomeric actinin (green), and Hoechst (cyan) in WT, untreated, and treated TTL p.G219S iPSCs-CMs with PTL (10 μM). Lower panel is the grayscale of the desmin of the same image of the upper panel. Levels of detyrosinated α-tubulin and desmin were identified based on intensity, and corrected total cell fluorescence was measured (n = 50–60 cells per group). Scale bar: 20 μm. (C) Immunoblots from the lysates from WT and TTL p.G219S iPSCs-CMs with and without with 10 μM PTL treatment, with quantifications below. (D) qPCR analysis of hypertrophic genes (fetal gene markers) in WT, treated, and untreated TTL p.G219S iPSC-CMs with PTL. Values are shown as mean ± SEM with each experiment performed in triplicate. Significance was evaluated by 1-way ANOVA with post hoc Tukey’s test. **P < 0.01, ***P < 0.001, and ****P < 0.0001.