IkBz is an inhibitor of the serine/threonine kinase and liquid-liquid phase separation that causes transition from adaptive cardiac hypertrophy to heart failure during pressure overload
Authors: Tanaka Y , Higashikuni Y , Liu W , Tanaka K , Hirata Y , Inuzuka R , Fukuda D , Kato M , Sata M , Komuro I 
Published: November 2023

Abstract

Background and Purpose
Inflammation contributes to both adaptive cardiac hypertrophy and heart failure. However, how inflammation causes transition from compensatory response to heart failure remains largely unknown. IkBz is a transcriptional modulator that changes gene expression pattern in the NF-kB system. IkBz does not have a DNA-binding domain and is suggested to exert its function through protein-protein interaction. IkBz is upregulated in the murine failing heart. This study was performed to elucidate the roles of IkBz and their mechanisms in the pathogenesis of heart failure.

Methods
Eight- to 12-week old wild-type (WT) and IκB hetero knockout (HKO) mice were used for animal experiments. Pressure overload was induced by transverse aortic constriction (TAC). Cardiac phenotype was assessed by echocardiographic and histological analyses. Bone marrow transplantation experiment was performed by injecting bone marrow cells isolated from donor femurs and tibias into 8-week-old mice. Neonatal rat ventricular myocytes (NRVMs) were used for in vitro experiments. Transcriptome was assessed in NRVMs treated with IkBz or control siRNA. Immunoprecipitation coupled with mass spectrometry (IP-MS) was performed by using NRVMs overexpressing FLAG-tag-labelled IkBz. Gene and protein expression levels were assessed by quantitative RT-PCR and Western blot.

Results
Pressure overload induced adaptive cardiac hypertrophy until 14 days after TAC and heart failure at 28 days after TAC in WT mice. TAC induced adaptive cardiac hypertrophy in HKO mice similarly to WT mice, whereas at 28 days after TAC HKO hearts showed preserved systolic function with attenuated left ventricular dilatation compared with WT hearts. Bone marrow transplantation experiment revealed that IkBz expressed in bone marrow-derived cells has little effect on cardiac phenotype after TAC. Transcriptome analysis showed that IkBz knockdown leads to upregulation of genes related to muscle contraction and extracellular matrix organization, which was confirmed by quantitative RT-PCR in heart samples of WT and HKO mice. IP-MS and IP/western blot demonstrated that IkBz binds to the epigenetic modulator and the serine/threonine kinase. IkBz inhibited liquid-liquid phase separation induced by the epigenetic modulator. In WT failing hearts, the serine/threonine kinase protein level was suppressed compared with sham-operated WT hearts, while the protein level of the serine/threonine kinase was higher in HKO hearts at 28 days after TAC compared with WT hearts. Pharmacological inhibition of the serine/threonine kinase in WT mice impaired cardiac compensatory response at 14 days after TAC.

Conclusion
Our results suggest that IkBz causes transition from adaptive cardiac hypertrophy to heart failure through inhibition of the serine/threonine kinase activity and liquid-liquid phase separation. IkBz might be a novel therapeutic target for heart failure.