The negative regulation of gene expression by microRNAs as key driver of inducers and repressors of cardiomyocyte differentiation

Abstract

Cardiac muscle damage-induced loss of cardiomyocytes (CMs) and dysfunction of the remaining ones leads to heart failure, which nowadays is the number one killer worldwide. Therapies fostering effective cardiac regeneration are the holy grail of cardiovascular research to stop the heart failure epidemic. The main goal of most myocardial regeneration protocols is the generation of new functional CMs through the differentiation of endogenous or exogenous cardiomyogenic cells. Understanding the cellular and molecular basis of cardiomyocyte commitment, specification, differentiation and maturation is needed to devise innovative approaches to replace the CMs lost after injury in the adult heart. The transcriptional regulation of CM differentiation is a highly conserved process that require sequential activation and/or repression of different genetic programs. Therefore, CM differentiation and specification have been depicted as a step-wise specific chemical and mechanical stimuli inducing complete myogenic commitment and cell-cycle exit. Yet, the demonstration that some microRNAs are sufficient to direct ESC differentiation into CMs and that four specific miRNAs reprogram fibroblasts into CMs show that CM differentiation must also involve negative regulatory instructions. Here, we review the mechanisms of CM differentiation during development and from regenerative stem cells with a focus on the involvement of microRNAs in the process, putting in perspective their negative gene regulation as a main modifier of effective CM regeneration in the adult heart.

Keywords: cardiac stem cells; microRNA; myogenesis; regeneration.

Figure 1. Overview of five major steps of the heart development

The schematic representation shows the stage specific main cardiac morphogens and molecular markers composing the regulatory signaling pathways of cardiomyocyte formation (commitment, specification, differentiation) during heart development. From left, cardiac crescent formation through mesoderm-derived cardiac progenitor activation, consisting of the first heart field (FHF) and second heart field (SHF) cardiac progenitors. Following is the linear heart tube formation and cardiomyocyte progenitors appearance and sequentially heart looping with initiation of chamber formation that ultimate with the formation of the septated heart. The last stage of the adult heart coincides with cardiomyocyte terminal differentiation.

Figure 2. Summary of miRNAs differentially expressed in five major steps of heart development

The schematic representation shows the most characterized miRNAs regulating cardiac development and cardiomyocyte formation.

Figure 3. Cardiac stem/progenitor cell derived cardiomyocyte formation*

(A) Schematic representation shows the differentiation protocol to derive beating cardiomyocytes from cloned CSCs in vitro. (B) Representative confocal image of mouse cloned CSCs-derived cardiospheres (CS) before myogenic differentiation induction (left), showing no cTnI or MF20 expressing cells and of functional cardiomyocytes derived from CS differentiation, showing a homogenous expression of cTnI (red) and MF20 (green). (C) Confocal microscopy examples of CSC-derived iCMs, neoCMs and adult CMs labeled with cTnI (red). (D) Heatmap showing qPCR analysis of main contractile genes in cardiosphere-derived CSCs (Actc1, Tnnt2, Myh7, Actc1, Myh6, Acta2, Cnn1) and cardiac transcription factors (Mef2c, Gata4, Nkx2.5, Hand 2 and Brachyury T) after myogenic differentiation. *Adapted from reference [78].