NUAK1 regulates contraction in vascular smooth muscle cells derived from abdominal aortic aneurysm patients

Rombouts, K. B.; van Merrienboer, T. A. R.; Henneman, A. A.; Knol, J. C.; Pham, T. V.; Piersma, S. R.; Jimenez, C. R.; Hordijk, P. L.; Bogunovic, N.; van der Velden, J.; Yeung, K. K.

doi:10.1101/2024.12.19.629332

bioRxiv · 10.1101/2024.12.19.629332

NUAK1 regulates contraction in vascular smooth muscle cells derived from abdominal aortic aneurysm patients

Rombouts, K. B.·van Merrienboer, T. A. R.·Henneman, A. A.·Knol, J. C.·Pham, T. V.·Piersma, S. R.·Jimenez, C. R.·Hordijk, P. L.·Bogunovic, N.·van der Velden, J.·Yeung, K. K.

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Abstract

AimAbdominal aortic aneurysms (AAA) are defined as a dilatation of the aortic wall. Impaired contractile ability of vascular smooth muscle cells (vSMC) is a hallmark of AAA. This study investigates the underlying mechanism of altered in vitro contractility of vSMC derived from AAA patients (AAA-SMC) compared to control vSMC (C-SMC). MethodsContractility of AAA-SMC (n=24) and C-SMC (n=8) was measured using Electric Cell- substrate Impedance Sensing. Large variability in AAA-SMC contraction was observed compared to C-SMC contraction, and AAA-SMC were therefore subdivided into low, intermediate (i.e. comparable to C-SMC contraction) and high contracting. To identify novel proteins involved in altered AAA-SMC contraction, a phosphoproteomic analysis was performed. ResultsThe proteomics data showed that Thrombospondin-1, PDZ and LIM domain protein 4 and ATPase plasma membrane Ca2+ transporting 1 expression correlated with vSMC contraction, but knockdown (KD) of these targets did not affect contraction. Next, the phosphoproteomics data identified NUAK family kinase 1 (NUAK1) as a potential regulator of contraction, since its kinase activity correlated with AAA-SMC contraction. NUAK1 regulates Myosin phosphatase targeting subunit 1 (MYPT1) activity by phosphorylation, as confirmed by the correlation between NUAK1 activity and phosphorylation levels of Ser445 and Ser910 on MYPT1. NUAK1 protein and RNA expression correlated with AAA-SMC contraction. Moreover, NUAK1 KD decreased contraction in AAA-SMC, combined with reduced phosphorylation levels of Myosin light chain (pMLC) and Vinculin gene expression, and increased F-actin cytoskeletal filament levels. ConclusionsNUAK1 regulates AAA-SMC contraction. Low NUAK1 expression decreased pMLC, potentially by higher MYPT1 activity, and subsequently reduced contraction. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=101 SRC="FIGDIR/small/629332v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@41c0f0org.highwire.dtl.DTLVardef@e1dd1borg.highwire.dtl.DTLVardef@7b36d7org.highwire.dtl.DTLVardef@14d65b5_HPS_FORMAT_FIGEXP M_FIG C_FIG Elements were modified from Servier Medical Art, licensed under a Creative Common Attribution 3.0 Generic License. https://smart.servier.com/; https://creativecommons.org/licenses/by/3.0/ HighlightsO_LIQuantification of in vitro vascular smooth muscle cells (vSMC) contractile capacity revealed large variation in contraction of vSMC derived from human AAA tissue (AAA- SMC) compared to vSMC derived from healthy aortic biopsies (C-SMC). C_LIO_LIPhosphoproteomic analysis identified NUAK family kinase 1 (NUAK1) as a novel regulator of contraction in AAA-SMC, since its kinase activity and expression levels correlated with AAA-SMC contraction. C_LIO_LINUAK1 regulates contraction in AAA-SMC by regulating Myosin phosphatase targeting subunit 1 (MYPT1) activity through phosphorylation, which subsequently affects phosphorylation levels of Myosin light chain (pMLC), and by affecting Vinculin expression and F-actin cytoskeletal filament levels. C_LIO_LIGaining more insight into NUAK1 function and mechanisms leading to aortic wall weakening can ultimately contribute to better AAA disease prediction and treatment. C_LI

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Rombouts, K. B., van Merrienboer, T. A. R., Henneman, A. A., Knol, J. C., Pham, T. V., Piersma, S. R., Jimenez, C. R., Hordijk, P. L., Bogunovic, N., van der Velden, J., Yeung, K. K.. 2024-12-20. NUAK1 regulates contraction in vascular smooth muscle cells derived from abdominal aortic aneurysm patients. https://doi.org/10.1101/2024.12.19.629332

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