Acute injury of the heart such as a myocardial infarction leads to the death of billions of cardiomyocytes (CMs). Adult human CMs are mostly terminal differentiated and unable to re-enter the cell cycle in a sufficient manner to regenerate the lost myocardium. Therefore, a permanent fibrotic scar is formed which leads to reduced heart function and eventually heart failure and death. Current medical therapies can only treat the symptoms but there is no cure available. An innovative and promising biomedical approach is to induce proliferation in spared CMs at the wound border to regenerate lost tissue and minimize scarring. To identify such novel pro-proliferative cues, we carried out in vivo high-throughput screening (HTS) of small bioactive molecule libraries. We used zebrafish embryos (Danio rerio) as a model, due to the almost unlimited number of transparent offspring, the ease of genome editing in this species and the availability of reporter lines that can be used as in vivo readout for CM number and proliferation. Moreover, adult zebrafish can regenerate the heart and resolve the fibrotic scar after injury due to the retained proliferative capacity of spared CMs. Regeneration involves dedifferentiation and re-entry into the cell cycle of spared CMs, which is accompanied by a more embryonic state of these CMs. Therefore, understanding what drives CM proliferation during development is crucial to find targets to boost adult CM proliferation and regeneration. Thus far we screened 345 compounds and identified several promising candidates. One of the most eye-catching hits was Pimozide (brand name Orap), a clinically relevant antipsychotic which is broadly used to treat diseases such as Tourette's syndrome or schizophrenia. Pimozide acts as an antagonist of the D2, D3, and D4 receptors (dopaminergic), the 5-HT7 receptor (serotonergic) as well as the t-type Ca2+ channels (CaV3.1 and CaV3.2). Treatment with Pimozide leads to an enlarged ventricle in zebrafish embryos as well as severe arrythmia. Further analysis of the phenotype demonstrated an increase of ventricular CM number and proliferation rate but no increase in cell size, suggesting Pimozide leads to hyperplasia rather than hypertrophy. Our current work focuses on a more precise characterization of the phenotype including ECG recordings and Ca2+ imaging to address the arrhythmia phenotype. Moreover, treatment with selective t-type Ca2+ channels, D2 receptor and HT7 receptor antagonists and transient knockdown of CaV3.1 and CaV3.2 channels will shed further light into the mechanisms underlaying the Pimozide phenotype.
