Constant Morez

Generation of de novo cardiomyocytes through viral over-expression of key transcription factors represents a highly promising strategy for cardiac muscle tissue regeneration. Although the feasibility of cell reprogramming has been proven possible both in vitro and in vivo, the efficiency of the process remains extremely low. Here, we report a chemical-free technique in which topographical cues, more specifically parallel microgrooves, enhance the directed differentiation of cardiac progenitors into cardiomyocyte-like cells. Using a lentivirus-mediated direct reprogramming strategy for expression of Myocardin, Tbx5, and Mef2c, we showed that the microgrooved substrate provokes an increase in histone H3 acetylation (AcH3), known to be a permissive environment for reprogramming by "stemness" factors, as well as stimulation of myocardin sumoylation, a post-translational modification essential to the transcriptional function of this key co-activator. These biochemical effects mimicked those of a pharmacological histone deacetylase inhibitor, valproic acid (VPA), and like VPA markedly augmented the expression of cardiomyocyte-specific proteins by the genetically engineered cells. No instructive effect was seen in cells unresponsive to VPA. In addition, the anisotropy resulting from parallel microgrooves induced cellular alignment, mimicking the native ventricular myocardium and augmenting sarcomere organization.

Direct reprogramming strategies for cell fate switching hold great promise for regenerative medicine. However, because of the strong stability provided by a cell's epigenome, the efficiency associated with such processes remains critically low. This study shows that the use of microgrooved substrates can dramatically alter a cell's epigenetic landscape. More specifically a significant disruption of the repressive heterochromatin is observed, characterised by a significant diminution of H3K27me3 and H3K9me3 levels and an increase in H3K4me3. In the specific case of cardiac direct programming through viral over-expression of key transcription factors, the use of these parallel microgrooves can significantly increase the number of induced cardiomyocyte-like cells generated. In addition microgrooved substrates can also stimulate histone acetylation, thereby increasing chromatin accessibility, and concomitantly stimulate sumoylation of the co-activator myocardin, thereby enhancing its cardiogenic capacity. Moreover, the alignment provided by the substrate can foster the establishment of organized sarcomeric structures, and therefore strengthen the maturation of the induced-cardiomyocytes. Lastly, the potential effect of the microgrooved substrate in neural progenitor differentiation is investigated. Interestingly, a strong epigenetic remodelling is also observed. However, apart from a notable induced histone hyper-acetylation, its characteristics are strikingly different, with notably a strong stimulation of the repressive H3K9me3 mark. Remarkably, neural progenitor differentiation triggered by the addition of soluble factors yielded significantly more neurons on the microgrooved substrates.