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Successful overexpression of wild-type inhibitor-2 of PP1 in cardiovascular cells

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Abstract

About half of the cardiac serine/threonine phosphatase activity is due to the activity of protein phosphatase type 1 (PP1). The activity of PP1 can be inhibited by an endogenous protein for which the expression inhibitor-2 (I-2) has been coined. We have previously described a transgenic mouse overexpressing a truncated form of I-2. Here, we have described and initially characterized several founders that overexpress the non-truncated (i.e., full length) I-2 in the mouse heart (TG) and compared them with non-transgenic littermates (WT). The founder with the highest overexpression of I-2 displayed under basal conditions no difference in contractile parameters (heart rate, developed tension, and its first derivate) compared to WT. The relative level of PP1 inhibition was similar in mice overexpressing the non-truncated as well as the truncated form of I-2. For comparison, we overexpressed I-2 by an adenoviral system in several cell lines (myocytes from a tumor-derived cell line (H9C2), neonatal rat cardiomyocytes, smooth muscle cells from rat aorta (A7R5)). We noted gene dosage-dependent staining for I-2 protein in infected cells together with reduced PP1 activity. Finally, I-2 expression in neonatal rat cardiomyocytes led to an increase of Ca2+ transients by about 60%. In summary, we achieved immunologically confirmed overexpression of wild-type I-2 in cardiovascular cells which was biochemically able to inhibit PP1 in the whole heart (using I-2 transgenic mice) as well as in isolated cells including cardiomyocytes (using I-2 coding virus) indirectly underscoring the importance of PP1 for cardiovascular function.

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Acknowledgements

The work was part of the thesis of Thorsten Krause.

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Authors and Affiliations

  1. Institut für Pharmakologie und Toxikologie, Westfälische Wilhelms-Universität, 48149, Münster, Germany

    Thorsten Krause, Stefanie Grote-Wessels, Felix Balzer, Peter Boknik, Uwe Kirchhefer, Frank U. Müller & Wilhelm Schmitz

  2. Institut für Pharmakologie und Toxikologie, Medizinische Fakultät, Martin-Luther-Universität Halle-Wittenberg, Magdeburger Str. 4, D-06112, Halle, Germany

    Ulrich Gergs & Joachim Neumann

  3. Institut für Hämatopathologie, 22547, Hamburg, Germany

    Igor B. Buchwalow

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  1. Thorsten Krause
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  2. Stefanie Grote-Wessels
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  3. Felix Balzer
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  4. Peter Boknik
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  5. Ulrich Gergs
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  6. Uwe Kirchhefer
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  7. Igor B. Buchwalow
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  8. Frank U. Müller
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  9. Wilhelm Schmitz
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  10. Joachim Neumann
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Contributions

F.U.M., W.S., and J.N. designed the research; T.K., S.G.-W., F.B., P.B., U.K., and I.B.B. performed research; T.K., S.G.-W., F.B., P.B., U.K., I.B.B., F.U.M., and U.G. analyzed data, U.G. and J.N. wrote the paper.

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Correspondence to Joachim Neumann.

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Krause, T., Grote-Wessels, S., Balzer, F. et al. Successful overexpression of wild-type inhibitor-2 of PP1 in cardiovascular cells. Naunyn-Schmiedeberg's Arch Pharmacol 391, 859–873 (2018). https://doi.org/10.1007/s00210-018-1515-3

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