STAT5bis a key effector ofNRG‐1/ERBB4‐mediated myocardial growth

Abstract

The growth factor Neuregulin‐1 (NRG‐1) regulates myocardial growth and is currently under clinical investigation as a treatment for heart failure. Here, we demonstrate in several in vitro and in vivo models that STAT5b mediates NRG‐1/EBBB4‐stimulated cardiomyocyte growth. Genetic and chemical disruption of the NRG‐1/ERBB4 pathway reduces STAT5b activation and transcription of STAT5b target genes Igf1, Myc, and Cdkn1a in murine cardiomyocytes. Loss of Stat5b also ablates NRG‐1‐induced cardiomyocyte hypertrophy. Dynamin‐2 is shown to control the cell surface localization of ERBB4 and chemical inhibition of Dynamin‐2 downregulates STAT5b activation and cardiomyocyte hypertrophy. In zebrafish embryos, Stat5 is activated during NRG‐1‐induced hyperplastic myocardial growth, and chemical inhibition of the Nrg‐1/Erbb4 pathway or Dynamin‐2 leads to loss of myocardial growth and Stat5 activation. Moreover, CRISPR/Cas9‐mediated knockdown of stat5b results in reduced myocardial growth and cardiac function. Finally, the NRG‐1/ERBB4/STAT5b signaling pathway is differentially regulated at mRNA and protein levels in the myocardium of patients with pathological cardiac hypertrophy as compared to control human subjects, consistent with a role of the NRG‐1/ERBB4/STAT5b pathway in myocardial growth.

Phosphoproteomic analysis reveals the diversity of signaling behind ErbB inhibitor-induced phenotypes

“…Differences in the phenotypes associated with the phosphorylation changes induced by the ErbB inhibitors were observed. As also previously reported (Paatero et al, 2019; Vaparanta et al, 2023), myocardial and neurological phenotypes were only associated with AG1478 and lapatinib treatment. Since the phosphoproteomic changes induced by the treatment of these inhibitors were markedly different, we wanted to explore if blocking the pathways differentially regulated by AG1478 and lapatinib would result in similar neurological and myocardial phenotypes in embryonic zebrafish to AG1478 or lapatinib treatment.…”

“…3A-B). To examine the correlation of previously (Vaparanta et al, 2023) and newly executed Western analyses with the results of the phosphoproteomic analysis, correlation analyses were performed. The intensity values of phosphosite-specific antibodies in Western analyses had a significant positive correlation with the intensity values of the phosphopeptides identified in the phosphoproteomic analysis and the enrichment scores of signaling pathways from the GSEAs that were performed on the phosphoproteomics data.…”

“…Despite these similarities, their effects on the global phosphoproteome were significantly different. These differences in effects on downstream signaling may explain also the observed differential biological effects (Paatero et al, 2019;Vaparanta et al, 2023). ErbB inhibitors differ in their efficacy and toxicity also in clinical setting (Dai et al, 2021), and one underlying cause could be differential effects on cell signaling.…”

“…Dunnett's multicomparison test was utilized for ANOVA post hoc analyses. C-D: Correlation of previously acquired (Vaparanta et al, 2023) and new Western analyses with the global phosphoproteome data at the phosphosite level (C) and with the GSEA enrichment scores of the pathways enriched in the phosphoproteome data (D). The mean LFQ-intensity values of the phosphopeptides containing the phosphorylated residue and densitometric measurements of the western analyses were normalized against the DMSO control and transformed to the Log2 scale.…”

“…Phenotypic analyses of zebrafish embryos exposed to ErbB inhibitors lapatinib, gefitinib, or AG1478 have shown differential phenotypes in cardiac biology and in the overall movement of the zebrafish embryos (Paatero et al, 2019; Vaparanta et al, 2023). Both the ventricle wall growth and the overall movement of the zebrafish embryos have been most strongly affected by AG1478 treatment and least with gefitinib treatment (Paatero et al, 2019; Vaparanta et al, 2023). This prompted us to analyze the signaling differences of these inhibitors in-depth using analysis of whole organism phosphoproteome using protein mass spectrometry.…”