Objectives: MicroRNAs (miRs) are small non-coding RNAs that regulate gene expression and modulate cardiac development and disease. MiR-128 is well known for its role in cancer but its role in cardiac development remains obscure. MiR-128 loss-of-function studies were conducted in vivo in zebrafish and in vitro by differentiating murine embryonic stem cells (ESCs).
Methods: Loss-of-function experiments in vivo were performed applying morpholino oligos (MOs) complementary to the miR-128 sequence. MOs were injected into zebrafish embryos (1-/2-cell stage) while the cardiac phenotype was analyzed 72 hours post fertilization (hpf). Loss-of-function experiments in vitro were conducted using LNA™ oligonucleotide probes (Fa. Exiqon) which inhibit mmu-miR-128–3p. Standard differentiation assays were performed with a murine ESC-line reporting cardiac progenitor cells by GFP-fluorescence which were quantified by flow cytometry. Beating rates of early cardiomyocytes were determined. MiR-128 expression and gene expression of typical cardiac marker genes were analyzed by qRT-PCR.
Results: MiR-128 downregulation in vivo revealed that zebrafish larvae (72hpf) displayed early signs of cardiac dysfunction including pericardial edema as well as significantly decreased beating rates and fractional shortening. MiR-128 deficient larvae further exhibited abnormalities in heart looping. During in vitro differentiation of murine ESCs significantly reduced beating rates of early cardiomyocytes were observed (after 7, 10, 14 days) in cells treated with miR-128 inhibiting LNA probes confirming the results of zebrafish experiments. MiR-128 was significantly downregulated by LNA probes on day 5, 7, 10, and 14 (>80%). MiR-128 inhibition increased Isl1 expression on day 5 and 7 during in vitro differentiation (upregulation of ~30% compared with controls). Isl1 is a direct target of miR-128 and an important player in cardiac development. Other cardiac genes, like Nkx2.5 or Mef2c, were not influenced by a knock-down of miR-128. Analyzing the three main cardiac lineages after two weeks of differentiation revealed no significant influence of miR-128 on gene expression of Tnnt2, αMHC, αActinin, CD31 or αSMA.
Conclusion: MiR-128 loss-of function studies in vivo and in vitro supported the hypothesis that miR-128 plays a role as modulator of early cardiac development. Downregulation of miR-128 led to cardiac dysfunction in zebrafish as well as changes during in vitro differentiation of murine ESCs.
