Meyer-Jens, M. | bioRxiv Science

bioRxiv2026

Neprilysin inhibition reduces microtubule detyrosination in cardiomyocytes through a cGMP-PRKG1-VASH1 axis

Microtubule detyrosination and re-tyrosination on the C-terminus of -tubulin are mediated by the vasohibin (VASH)-small vasohibin-binding protein (SVBP) complex and tubulin tyrosine ligase (TTL), respectively. Elevated levels of detyrosinated -tubulin (dTyr-tub) are observed in heart failure, and reducing this modification improves cardiac function, suggesting that clinically used heart failure therapies may modulate microtubule detyrosination. We investigated whether sacubitrilat and valsartan, the active components of the angiotensin receptor-neprilysin inhibitor LCZ696, influence dTyr-tub levels in endothelin-1 (ET1)-induced hypertrophy in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). While both sacubitrilat and valsartan prevented hypertrophy, only sacubitrilat prevented ET1-induced dTyr-tub accumulation. RNA sequencing revealed that sacubitrilat normalized several ET1-induced dysregulated pathways. Sacubitrilat slightly increased cyclic guanosine 3,5-monophosphate (cGMP) levels and lowered dTyr-tub, whereas inhibition or knockdown of the cGMP-dependent protein kinase 1 (PRKG1) increased dTyr-tub level. Mechanistically, PRKG1 alpha phosphorylated native VASH1. Incubation of microtubules with the VASH1-SVBP complex containing wild-type VASH1 increased detyrosination, while incubation of the complex containing a VASH1 phosphomimic, in which seven C-terminal serine residues were mutated to glutamate (VASH1-7E) did not. Consistently, overexpression of VASH1-7E gave rise to lower dTyr-tub level than overexpression of a non-phosphorylatable form of VASH1 (VASH1-7A) in hiPSC-CMs deficient in VASH1. In conclusion, these findings identify a cGMP-PRKG1-VASH1 signaling axis that reduces microtubule detyrosination in cardiomyocytes. Our work provides mechanistic insight into how neprilysin inhibition may contribute to therapeutic benefit in heart failure. One Sentence SummaryWe establish a neprilysin-cGMP-PRKG1-VASH1 signaling axis that reduces microtubule detyrosination in cardiomyocytes.

cell biology↗

bioRxiv2024

Ruxolitinib clears CRYAB p.Arg120Gly aggregates through the ubiquitin-proteasome system

RationaleProtein accumulation is a hallmark of many neurodegenerative and muscular diseases. Desmin-related (cardio-) myopathy (DRM), a well-studied model for cardiac muscle protein accumulation, is an autosomal dominant-inherited disease presenting with progressive muscle weakness, reduced quality of life, and shortened life span. To date, DRM patients are treated symptomatically and there is no causal treatment available. Independent of the genetic cause, most DRM patients display intracellular accumulation of desmin and its chaperone B-crystallin (CRYAB). We previously conducted an unbiased high-throughput screen to identify novel effectors that reduce cardiomyocyte aggregate levels and found that downregulation of Janus kinase 1 (JAK1) resulted in lower aggregate load in neonatal mouse cardiomyocytes. ObjectiveIn this study, we tested if the approved JAK inhibitor ruxolitinib ameliorates the disease phenotype in rodent and human CRYAB p.Arg120Gly DRM models. Methods and ResultsWe found that the mRNA levels of Jak1 and Stat3 were higher than any other JAK-signal transducer and activator of transcription (STAT) family members in neonatal rat ventricular myocytes (NRVMs) and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). The approved JAK1/2 inhibitor ruxolitinib and the JAK1 inhibitors solcitinib, upadacitinib, and filgotinib prevented accumulation of and cleared pre-existing CRYAB p.Arg120Gly protein aggregates in NRVMs and hiPSC-CMs. Importantly, the knockdown of Jak1 and Stat3, but not Jak2 resulted in fewer aggregates. Moreover, ruxolitinib, Jak1 or Stat3 siRNA treatment enhanced the ubiquitin-proteasome system (UPS)-mediated degradation. Blocking UPS function blunted the effect of ruxolitinib or Jak1 siRNA on CRYAB p.Arg120Gly accumulation. RNAseq of NRVMs treated with Jak1 siRNA extracts revealed higher gene expression of important muscle E3 ubiquitinating enzymes. Knockdown of the E3 ligase Asb2 (Ankyrin Repeat And SOCS Box Protein 2) abolished the effect of ruxolitinib on CRYAB p.Arg120Gly aggregates. In DRM mice, phospho-STAT3 levels were markedly higher than in non-transgenic (NTG) mice with age. Ruxolitinib treatment or Jak1 knockout prevented cardiac dysfunction and reduced CRYAB p.Arg120Gly aggregate load in DRM mice. ConclusionIn this study, we uncovered the previously unknown effect of the approved drug ruxolitinib to enhance UPS-mediated degradation and prevent protein aggregates in cardiomyocytes.

pharmacology and toxicology↗