Transcriptome analysis of conditionally immortalized atrial myocytes: identification of a novel atrium-enriched protein involved in sarcomere assembly and maintenance

European Heart Journal, vol. 42

Abstract

Abstract Background Heart development relies on the tight spatiotemporal control of cardiac gene expression. Genes involved in these processes have been identified using mainly (transgenic) animals models and pluripotent stem cell-derived cardiomyocytes (CMs). Recently, the repertoire of cardiomyocyte differentiation models has been expanded with iAM-1, a monoclonal cell line of conditionally immortalized neonatal rat atrial myocytes (NRAMs) which allows toggling between proliferative and differentiated (i.e. excitable and contractile) phenotypes in a synchronized and homogenous manner. Purpose To identify and characterize (lowly expressed) genes with an as-of-yet uncharacterized role in cardiomyocyte differentiation, dedifferentiation and proliferation by exploiting the unique properties of conditionally immortalized NRAMs (iAMs). Methods and results RNA sequencing was performed during a full cycle of iAM-1 differentiation and subsequent dedifferentiation, identifying ±13,000 transcripts, of which the dynamic expressional changes during cardiomyogenic differentiation in most cases opposed those during dedifferentiation. Among the genes whose expression increased during differentiation and decreased during dedifferentiation were many genes with a known (lineage-specific) role in cardiac muscle formation, thereby confirming the relevance of iAMs as cardiomyogenic differentiation model. Filtering for cardiomyocyte-enriched low abundancy transcripts, resulted in the identification of an uncharacterized protein, which is highly conserved among Nephrozoa and up- and downregulated during cardiomyocyte differentiation and dedifferentiation, respectively. In neonatal and adult rats, this protein is muscle-specific, highly atrium-enriched and localized around the C-zone of cardiac sarcomeres. Lentiviral shRNA-mediated knockdown resulted in loss of sarcomeric organization in both NRAMs and iAMs. Neither knockdown nor overexpression of this protein affected the electrophysiological properties of differentiated iAM monolayers. Conclusions iAM-1 cells offer a relevant model to identify and characterize novel (low abundancy) genes involved in cardiomyocyte (de)differentiation as exemplified by the identification a novel uncharacterized protein that is muscle-specific, highly atrium-enriched, localized around the C-zone of cardiac sarcomeres and plays a specific role in atrial sarcomerigenesis. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Netherlands Organisation for Health Research and Development (ZonMw) Leiden Regenerative Medicine Platform Holding project with number (LRMPH) Figure 1. (A) Experimental setup. At the indicated timepoints iAM-1 cells were fixed for immunostaining and RNA extraction for transcriptome analysis. (B) Immunochemical staining of iAM-1 cells for the proliferation marker Ki-67 and the Z-line marker sarcomeric α-actinin. (C & D) Immunohistological double stainings of longitudinal sections of neonatal rat hearts for the uncharacterized protein (GOI 1) and the sarcomeric protein cardiac troponin I (TNNI3). LA, left atrium; RA, right atrium; LV, left ventricle; RV, right ventricle. Scale bar, 250 μm.

Authors 12

1. Pim R.R. van Gorp Aachen

   Leiden University · Leiden University Medical Center

   Affiliation as printed

   Leiden University Medical Centre, Leiden, Netherlands (The)

2. Juan Zhang Aachen

   Leiden University · Leiden University Medical Center

   Affiliation as printed

   Leiden University Medical Centre, Leiden, Netherlands (The)

3. J Liu Aachen

   Leiden University · Leiden University Medical Center

   Affiliation as printed

   Leiden University Medical Centre, Leiden, Netherlands (The)

4. Roula Tsonaka Aachen

   Leiden University · Leiden University Medical Center

   Affiliation as printed

   Leiden University Medical Centre, Leiden, Netherlands (The)

5. Hailiang Mei Aachen

   Leiden University · Leiden University Medical Center

   Affiliation as printed

   Leiden University Medical Centre, Leiden, Netherlands (The)

6. S O Dekker Aachen

   Leiden University · Leiden University Medical Center

   Affiliation as printed

   Leiden University Medical Centre, Leiden, Netherlands (The)

7. C I Bart Aachen

   Leiden University · Leiden University Medical Center

   Affiliation as printed

   Leiden University Medical Centre, Leiden, Netherlands (The)

8. Martin Jan Schalij Aachen

   Leiden University · Leiden University Medical Center

   Affiliation as printed

   Leiden University Medical Centre, Leiden, Netherlands (The)

9. Tim De Coster Aachen

   Leiden University · Leiden University Medical Center

   Affiliation as printed

   Leiden University Medical Centre, Leiden, Netherlands (The)

10. D A Atsma Aachen

    Leiden University · Leiden University Medical Center

    Affiliation as printed

    Leiden University Medical Centre, Leiden, Netherlands (The)

11. Daniël A. Pijnappels Aachen

    Leiden University · Leiden University Medical Center

    Affiliation as printed

    Leiden University Medical Centre, Leiden, Netherlands (The)

12. Antoine A.F. de Vries Aachen

    Leiden University · Leiden University Medical Center

    Affiliation as printed

    Leiden University Medical Centre, Leiden, Netherlands (The)