Can human heart regenerate? A new possible strategy

The World Health Organization reports that cardiovascular diseases (CVDs) are the number one cause of death globally, more than 30% of all deaths worldwide [1]. 85% of these cases arise out of heart failure. Lack of a successful pharmacological strategy worsens heart reluctance to regeneration. For this reason, there is a great interest in cardiac drug discovery. In 2019 the Australian research group headed by Hudson and Porrello published a scientific article in the Cell Stem Cell journal regarding their last research on this field [2].

In this study, Mills and collaborators employed 3D human cardiac cell cultures known as human cardiac organoids (hCO) to improve drug screening pipeline. This experimental system is more stable and effective than traditional 2D cell culture and allows a better comprehension of the molecular mechanisms acting in cells and tissues [3]. This is true especially for cardiovascular drug discovery and development [4]. Cardiac proliferation was assessed following different drug treatments. ~5000 compounds were initially screened in 2D human pluripotent stem cell-derived cardiomyocytes and then in immature proliferative hCOs, followed by validation in mature, fully differentiated hCOs. 105 small molecules were finally screened on hCO owing to their pro-regenerative potential. Noteworthy was the fact that some compounds, which induced proliferation in 2D, failed to induce it in the immature hCOs. If on one hand the pharmacological treatment triggers proliferation, on the other one this signal alters cardiomyocyte’s functionalities leading to an increased risk of arrhythmogenesis. As a consequence, the authors highlight the importance of screening compounds that promote proliferation without interfering with contractile parameters. From the list of 105 molecules, compound 3 and compound 65 were the two boosting proliferation in mature hCOs without adversely affecting contractile properties. In order to understand how these compounds drove the proliferation status, the authors performed RNA-sequencing (RNAseq) and single-organoid quantitative proteomics. Thanks to this analysis it was acknowledged that compound 3 regulated a mitogen-activated protein kinase 14 (MAPK 14) network, while upregulating the transcription of many cell-cycle controllers. Compound 65 regulated TGF-β and BMP networks, while inhibiting the transcription of the cell cycle inhibitor CDKN2B (Fig.2). Interestingly, compound 65 activated multiple enzymes in the mevalonate pathway (that is essential for the upstream of the cholesterol biosynthetic pathway) (Fig.2). The molecular profiles of the cells treated with the compounds 3 and 65 were compared to characterize the proliferation signature underlying the proliferation process. Integration of the data from cells treated with GSK3 and MST1 inhibitors, known to be cell cycle activators, expanded the molecular insights for compound 3 and 65. This analysis revealed a core overlap of 11 proteins, with 7 proteins belonging to cell-cycle networks, 2 proteins in the mevalonate / cholesterol pathway, and 2 nuclear transporters. This pharmacological approach indicates that both a cell-cycle and the mevalonate pathway have to be simultaneously active for cell-cycle induction in cardiomyocytes. This evidence underlined the role of the mevalonate pathway as a requirement in active cardiomyocyte proliferation.

Further proof of mevalonate implication came from another pharmacological assay. The research group proceeded by blocking the cholesterol biosynthesis pathway with simvastatin, a HMGCR inhibitor. Under these conditions, proliferation of cardiomyocytes was reduced as well as their size was affected. The presence of simvastatin also abolished the regenerative potential of compounds 3 and 65. Finally the experiment was brought from an in vitro to an in vivo system. Post-natal mice were injected with both compounds and an increase of DNA synthesis in the mouse cardiomyocytes was observed. The addition of simvastatin blocked the DNA synthesis confirming the importance of the mevalonate pathway. From a molecular point of view, additional experiments demonstrated how proliferation was dependent on YAP1, a known master regulator of cardiomyocyte proliferation.