Small molecule screen identifies inducers of mammalian cardiomyocyte proliferation

Abstract

Cardiovascular diseases remain among the leading cause of deaths worldwide accounting for about 40% of all postnatal deaths. Though the current therapies offer a temporary cure, owing to their limitations, there is still a need to develop alternative therapies. Adult mammalian hearts cannot regenerate as zebrafish, newt, and 1-day-old mice hearts do after an injury. Heart regeneration occurs in these animals by proliferation of pre-existing cardiomyocytes. During mammalian heart development the heart grows through cardiomyocyte proliferation, which rapidly ceases after birth. Therefore, we were interested in inducing postnatal cardiomyocyte proliferation in mammals by utilizing a chemical biology approach.We established a cardiomyocyte-specific live fluorescent-based fucci technique to screen small molecules to induce cardiomyocyte proliferation. We validated the platform first with known inducers and then screened two small molecule libraries containing modulators of nuclear receptor and epigenetic signatures in neonatal cardiomyocytes. Positive hits were further analyzed for their capacity to induce cell cycle re-entry and mitosis in P3 rat cardiomyocytes by for example BrdU incorporation and histone H3 phosphorylation analyses. We found that carbacyclin, an agonist of PPARdelta, induces robust cell cycle re- entry of cardiomyocytes.Carbacyclin in neonatal cardiomyocytes induced DNA synthesis, mitosis and cytokinesis but not binucleation. The effect of carbacyclin on cardiomyocytes was more potent than 10% serum (induces rather binucleation than proliferation). In fact it was comparable to the effect of FGF1+p38i, which has previously been shown to induce cardiomyocyte mitosis, to reduce scarring and to rescue function after Myocardial infarction (MI).It has been shown that 1-day-old, but not 7-day-old, mice can regenerate their heart. Therefore, we tested whether carbacyclin can induce P8 and adult cardiomyocytes cell cycle re-entry. Carbacyclin induced both BrdU incorporation and mitosis in P8 and adult cardiomyocytes in vitro. A closer analysis of P8 and adult cardiomyocytes revealed that the majority of cardiomyocytes that entered the cell cycle were mono-nucleated. Similar observations have been made in Neuregulin-treated cardiomyocytes. This result might be important for human therapy, as a majority of human cardiomyocytes is mono-nucleated. These data suggest that different populations of cardiomyocytes with different proliferative potential exist.The PPARs are best known as regulators of energy homeostasis and have many beneficial physiological functions. The cardiomyocyte- restricted PPAR delta deletion causes cardiac dysfunction, hypertrophy, and lipotoxic cardiomyopathy. In contrast, overexpression of PPAR delta has been shown to be protective to ischemia/reperfusion (I/R) injury and TAC-induced pressure overload. We found cardiomyocyte-restricted inducible dominant-active PPAR delta in vivo induces adult cardiomyocyte mitosis.PPARdelta increases fatty acid synthesis and catabolism as well as glucose and fatty acid transport and glycolysis, which might support cardiomyocyte cell cycle re-entry. Recently, it has been shown that deletion of Meis1, which is known to shift energy metabolism from glycolysis to oxidative phosphorylation, is sufficient to extend the proliferation potential of postnatal cardiomyocytes beyond day 1 to day. Thus, changes or shift in energy metabolism might be a way to overcome the cell cycle arrest in cardiomyocytes.We have shown that activation of PPAR delta; by carbacyclin induces the PPARdelta/PDK1/p308Akt/pGSK3-beta/beta-catenin pathway. This data coincides with recently published data that PPAR delta governs Wnt signaling and bone turnover by activation of direct interaction with B-catenin and activation of Lrp5. The stabilization and translocation of beta-catenin to nucleus is important to induce cardiomyocyte proliferation and we found enhanced carbacyclin-induced cardiomyocyte proliferation in the presence of BIO.Taken together, our data indicate that the fucci system can be utilized as screening platform for chemical libraries, natural compound libraries and whole genome siRNA libraries to identify critical signaling pathways, druggable targets and compounds to promote cardiac regeneration based on cardiomyocyte proliferation; if this is possible. The presented screening system eliminates the need of laborious and expensive techniques like immunofluorescence staining, incorporation of nucleotide analogues or cell count and metabolic assays. Our initial screen has identified carbacyclin as a potential inducer of cardiomyocyte proliferation and its target PPAR delta as a new regulator of cardiomyocyte proliferation and as a new target for regenerative heart therapies. To our knowledge, this is the first live-imaging screening tool to identify inducers of cardiomyocyte proliferation and the first description that carbacyclin induces cardiomyocyte proliferation. We have dissected the carbacyclin-induced PPARdelta/PDK1/p308Akt/pGSK3 beta/beta-catenin signaling pathway. Our data suggest moreover that carbacyclin induces cell cycle re- entry specifically in mono-nucleated cardiomyocytes in vitro and that overexpression of dominant-active PPARdelta induces adult cardiomyocyte mitosis in vivo. Herz-Kreislauf-Erkrankungen gehören nach wie vor zu den häufigsten Ursachen für Todesfälle weltweit, die rund 40% aller postnatalen Todesfälle zugrunde liegen. Gegenwärtige Therapien bieten in der Regel nur eine vorübergehende symptomatische Behandlung, da sie die primäre Ursache der meisten Herzerkrankungen, den Verlust an Herzmuskelzellen, nicht beheben können. Aus diesem Grund gibt es die Notwendigkeit neue Therapien zu entwickeln. Adulte Säugetierherzen können nicht wie der Zebrafisch, der Lurch bzw. 1 Tage-alte Mäuse ihr Herz nach eine Verletzung regenerieren. Die Herzregeneration erfolgt bei diesen Tieren durch die Proliferation von bereits vorhandenen Herzmuskelzellen. Zudem wächst das Herz während der Säugertierentwicklung ebenfalls durch die Proliferation von Herzmuskelzellen. Aus einem unbekannten Grund hören die Zellen jedoch kurz nach der Geburt auf zu proliferieren. Deshalb sind wir daran interessiert mittels eines chemisch-biologischen Ansatzes neue Induktoren der Herzmuskelzellproliferation zu identifizieren.Hier haben wir eine Herzmuskelzell-spezifische Fluoreszenz-basierte Methode entwickelt, um kleine chemische Moleküle effizient auf ihre Fähigkeit Herzmuskelzellproliferation zu induzieren zu untersuchen. Die Methode wurde zunächst mit bekannten Induktoren validiert und dann genutzt, um zwei Molekül-Bibliotheken (1: Modulatoren nukleärer Rezeptoren; 2: Epigenetische Modulatoren) nach Kandidaten zu untersuchen. Identifizierte Kandidaten wurden weiter auf ihre Fähigkeit analysiert, den Zellzyklus in Herzmuskelzellen zu reaktivieren (z.B. BrdU-Einbau), Mitose zu induzieren (Histon H3 Phosphorylierung). In dieser Arbeit wurde Carbacyclin, ein Agonist von PPARdelta, als robuster Aktivator des Zellzyklus in Herzmuskelzellen identifiziert.