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

Sarah C Hoelscher,Lutz Hein,Giovanni Cuda,Markus Krane,Heribert Schunkert,Gianluca Santamaria,Harald Lahm,Ralf Gilsbach,Martina Dreßen,Irina Neb,Zouhair Aherrahrou,Jeanette Erdmann,Theresia Stich,Rüdiger Lange,Zhong Zhang,Stefanie A Doppler,David Hassel,Anne Diehm

doi:10.3390/ijms21031158

Abstract

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 Nkx2.5 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 Isl1, Sfrp5, and Hcn4 (cardiac transcription factors) but reduced Irx4 at the onset of cardiogenesis, (2) upregulated Isl1-positive CPCs, whereas NkxCE-positive CPCs were downregulated, and (3) increased the expression of the ventricular cardiomyocyte marker Myl2 accompanied by a reduced beating frequency of early cardiomyocytes. Overexpression of miR-128a (4) diminished the expression of Isl1, Sfrp5, Nkx2.5, and Mef2c, but increased Irx4, (5) enhanced NkxCE-positive CPCs, and (6) favored nodal-like cardiomyocytes (Tnnt2+, Myh6+, Shox2+) 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.

Highlights

More than 2500 microRNAs have been detected in humans [1] and it is predicted that most mammalian mRNAs are regulated by at least one miR [2]
The analyzed cardiac progenitor cells (CPCs) are characterized by an active 2.1 kb Nkx2.5 cardiac enhancer (NkxCE) element [8,14] and are able to develop in all major cardiac cell types, namely cardiomyocytes (CMs), endothelial cells (ECs), smooth muscle cells (SMCs), and cardiac fibroblasts (CFs) [15]
For miR profiling, GFP-positive CPCs were isolated from transgenic NkxCE-GFP mouse embryos at E9.5 and from one week in vitro differentiated murine embryonic stem cells (ESCs) with the same transgenic background using flow cytometry (Figure 1C, Figure S1A,B)

Summary

Introduction

More than 2500 microRNAs (miRs) have been detected in humans [1] and it is predicted that most mammalian mRNAs are regulated by at least one miR [2]. MiRs seem to be involved in most physiological and pathophysiological processes in the body. Given this complexity, a complete picture of miR-dependent regulations in development, homeostasis, and disease is still emerging, and the specific roles of several miRs remain elusive. The heart is the first and most important organ to develop during embryogenesis. Cardiogenesis relies on a tightly regulated and synchronized network of growth factors, cardiac transcription factors, as well as non-coding RNAs. Dozens of miRs have already been described as indispensable players in cardiac development [3,4]. Most steps in the developing heart including cardiovascular lineage differentiation and cardiac cell proliferation involve regulatory actions of miRs [5,6]. It is indispensable to precisely understand how miRs influence cardiac development

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Discussion

Conclusion