Abstract 685 Deciphering cardiac CITED4 signaling by analysis of nascent proteomics and secretomics

Introduction

Neuregulin-1 (NRG1) is a cardioactive growth factor that acts on ErbB2/4 receptor tyrosine kinases and signals via PI3K/Akt pathway. It is associated with stimulation of cardiomyocyte proliferation and physiological cardiac hypertrophy. Previous results show that Neuregulin-1 induces the expression of transcriptional co-activator Cited4 (C4). Cardiac C4 is upregulated in both physiological cardiac growth and is necessary to prevent adverse remodeling in both settings in vivo. However, the exact mechanism of how Cited4 regulates Neuregulin-1-signaling is still largely unknown.

Results

We found that cell size as well as viability were significantly reduced in neonatal rat ventricular cardiomyocytes (NRVM) with C4 depletion alone relative to control siRNA treated cells (p<0.05, n=3-5). Furthermore, we confirmed dose-dependent C4 mRNA expression in response to NRG1 stimulation in NRVM (p<0.05, n=4). We then used a novel innovative technique combining pulsed-SILAC labeling, click-chemistry and mass spectrometry, allowing specific capturing of the immediate changes in the proteome and secretome after NRG1 stimulation in NRVMs. Computational analysis and comparison of the nascent proteome after 12h and 24h of NRG1 stimulation in C4-knockdown and control NRVM revealed that C4 significantly regulates proteins responsible for translation, RNA processing and energy derivation. Consistent with previously observed development of cardiac fibrosis in C4 knockout mice in vivo, we found significant upregulation of TGFb2 and TGFb2-related proteins in C4-knockdown NRVMs. We next confirmed the upregulation of TGFb2 and its downstream effectors in samples of C4-knockdown and control NRVM using Western Blotting. Additionally, we found TGFb2 secretion increased from C4 knockdown cells with NRG1 stimulation by analysis of the nascent secretome.

Conclusion

In conclusion, nascent proteomics and secretomics help to elucidate the complex C4-dependent intra- and extracellular NRG-signaling. Interestingly, we found a direct mechanistic link between C4- and TGFb-signaling. Further investigations will focus on further deciphering C4-depentend TGFb regulation, for example through protein-interactions. Ultimately, we aim to identify novel and targetable cardioprotective pathways in the heart and a better understanding of C4-dependent signaling may be an important contribution.

Clin Res Cardiol (2023). https://doi.org/10.1007/s00392-023-02180-w
Deciphering cardiac CITED4 signaling by analysis of nascent proteomics and secretomics
V. Knobloch¹, P. Schlegel¹, L. Lust¹, K. Aljakouch², C. Heß¹, T. Kuhn³, V. J. Bezzerides⁴, S. Mittag¹, M. Roshan¹, N. Frey³, J. Krijgsveld², C. Lerchenmüller³
¹Innere Medizin III, Kardiologie, Universitätsklinikum Heidelberg, Heidelberg; ²DKFZ, Heidelberg; ³Klinik für Innere Med. III, Kardiologie, Angiologie u. Pneumologie, Universitätsklinikum Heidelberg, Heidelberg; ⁴Boston Children´s Hospital, Boston, US;
Introduction Neuregulin-1 (NRG1) is a cardioactive growth factor that acts on ErbB2/4 receptor tyrosine kinases and signals via PI3K/Akt pathway. It is associated with stimulation of cardiomyocyte proliferation and physiological cardiac hypertrophy. Previous results show that Neuregulin-1 induces the expression of transcriptional co-activator Cited4 (C4). Cardiac C4 is upregulated in both physiological cardiac growth and is necessary to prevent adverse remodeling in both settings in vivo. However, the exact mechanism of how Cited4 regulates Neuregulin-1-signaling is still largely unknown. Results We found that cell size as well as viability were significantly reduced in neonatal rat ventricular cardiomyocytes (NRVM) with C4 depletion alone relative to control siRNA treated cells (p<0.05, n=3-5). Furthermore, we confirmed dose-dependent C4 mRNA expression in response to NRG1 stimulation in NRVM (p<0.05, n=4). We then used a novel innovative technique combining pulsed-SILAC labeling, click-chemistry and mass spectrometry, allowing specific capturing of the immediate changes in the proteome and secretome after NRG1 stimulation in NRVMs. Computational analysis and comparison of the nascent proteome after 12h and 24h of NRG1 stimulation in C4-knockdown and control NRVM revealed that C4 significantly regulates proteins responsible for translation, RNA processing and energy derivation. Consistent with previously observed development of cardiac fibrosis in C4 knockout mice in vivo, we found significant upregulation of TGFb2 and TGFb2-related proteins in C4-knockdown NRVMs. We next confirmed the upregulation of TGFb2 and its downstream effectors in samples of C4-knockdown and control NRVM using Western Blotting. Additionally, we found TGFb2 secretion increased from C4 knockdown cells with NRG1 stimulation by analysis of the nascent secretome. Conclusion In conclusion, nascent proteomics and secretomics help to elucidate the complex C4-dependent intra- and extracellular NRG-signaling. Interestingly, we found a direct mechanistic link between C4- and TGFb-signaling. Further investigations will focus on further deciphering C4-depentend TGFb regulation, for example through protein-interactions. Ultimately, we aim to identify novel and targetable cardioprotective pathways in the heart and a better understanding of C4-dependent signaling may be an important contribution.
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