miR-128a Acts as a Regulator in Cardiac Development by Modulating Differentiation of Cardiac Progenitor Cell Populations

• Main Authors: Sarah C. Hoelscher, Theresia Stich, Anne Diehm, Harald Lahm, Martina Dreßen, Zhong Zhang, Irina Neb, Zouhair Aherrahrou, Jeanette Erdmann, Heribert Schunkert, Gianluca Santamaria, Giovanni Cuda, Ralf Gilsbach, Lutz Hein, Rüdiger Lange, David Hassel, Markus Krane, Stefanie A. Doppler
• Format: Article
• Language: English
• Published: MDPI AG 2020-02-01
• Series: International Journal of Molecular Sciences
• Subjects: microrna; mir-128; cardiac development; cardiac progenitor cells; <i>nkx2.5</i> cardiac enhancer
• Online Access: https://www.mdpi.com/1422-0067/21/3/1158

MicroRNAs (miRs) appear to be major, yet poorly understood players in regulatory networks guiding cardiogenesis. We sought to identify miRs with unknown functions during cardiogenesis analyzing the miR-profile of multipotent <i>Nkx2.5</i> enhancer cardiac progenitor cells (NkxCE-CPCs). Besides well-known candidates such as miR-1, we found about 40 miRs that were highly enriched in NkxCE-CPCs, four of which were chosen for further analysis. Knockdown in zebrafish revealed that only miR-128a affected cardiac development and function robustly. For a detailed analysis, loss-of-function and gain-of-function experiments were performed during in vitro differentiations of transgenic murine pluripotent stem cells. MiR-128a knockdown (1) increased <i>Isl1</i>, <i>Sfrp5</i>, and <i>Hcn4</i> (cardiac transcription factors) but reduced <i>Irx4</i> at the onset of cardiogenesis, (2) upregulated <i>Isl1</i>-positive CPCs, whereas NkxCE-positive CPCs were downregulated, and (3) increased the expression of the ventricular cardiomyocyte marker <i>Myl2</i> accompanied by a reduced beating frequency of early cardiomyocytes. Overexpression of miR-128a (4) diminished the expression of <i>Isl1</i>, <i>Sfrp5</i>, <i>Nkx2.5</i>, and <i>Mef2c</i>, but increased <i>Irx4</i>, (5) enhanced NkxCE-positive CPCs, and (6) favored nodal-like cardiomyocytes (<i>Tnnt2</i>⁺, <i>Myh6</i>⁺, <i>Shox2</i>⁺) accompanied by increased beating frequencies. In summary, we demonstrated that miR-128a plays a so-far unknown role in early heart development by affecting the timing of CPC differentiation into various cardiomyocyte subtypes.