Evaluation of cGMP-driven post-translational microtubule modifications in human cardiomyocytes

Abstract

Increased microtubule detyrosination is a hallmark of heart failure and has been consistently observed in various in vitro and in vivo models. As heart failure prevalence rises worldwide, understanding the disease mechanisms and developing new therapeutic options are needed. Previous studies showed that reducing microtubule detyrosination improved cardiomyocyte contractility (Caporizzo et al., 2020; C. Y. Chen et al., 2020; Eaton et al., 2023; Robison et al., 2016). The heart failure drug LCZ696 (sacubitril/valsartan) outperforms standard therapy, but its effect on microtubule detyrosination/re-tyrosination has not been explored.

The main aim of my PhD Thesis was therefore to evaluate the impact of LCZ696 in vitro and to decipher the cellular pathway involved.

We established an endothelin 1-induced hypertrophy model with human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). These cells exhibited a twofold increase in cell area and volume and a >1-5-fold increase in microtubule detyrosination, alongside typical transcriptomic and proteomic alterations. Cellular hypertrophy was prevented by sacubitrilat (sac), valsartan (val) or the combination of both, but only sac alone prevented the molecular and detyrosination changes. Reduction in microtubule detyrosination was also measured in mice subjected to transverse aortic constriction treated with LCZ696.

We hypothesized that sac reduced microtubule detyrosination via the cGMP-PRKG1 signaling. PRKG1 knockdown or inhibition increased detyrosination in hiPSC-CMs, while natriuretic peptides and sac activated PRKG1, indicated by VASP phosphorylation at serine 239. In hiPSC-CMs lacking the ability to re-tyrosinate microtubules, atrial natriuretic peptide increased microtubule tyrosination, suggesting PRKG1 inhibits the detyrosinase vasohibin 1 (VASH1) by phosphorylation. A VASH inhibitor produced the same effect, supporting the mechanism. Collaborators identified seven potential PRKG1A phosphorylation sites in the VASH1 C-terminus. Mutation of these sites to mimic phosphorylation reduced enzyme activity after transduction in VASH1-deficient hiPSC-CMs.

Since hiPSC-CMs deficient in vasohibin activity still exhibited about 20% of microtubule detyrosination (Pietsch et al., 2024), the last part of this PhD thesis was to assess the role of the newly identified detyrosinase MATCAP1. We successfully established a MATCAP1-deficient hiPSC line, which passed all implied quality control steps. In hiPSC-derived 2D culture of CMs or 3D engineered heart tissues, MACAP1 knockout caused extensive proteomic changes affecting structural and metabolic pathways and reduction in microtubule detyrosination in the EHTs.

Overall, our results show that microtubule detyrosination is a key driver of cardiomyocyte hypertrophy and highlight cGMP-PRKG1 pathway as a potential therapeutic target to improve heart failure outcome.