Abstract Body: Introduction: Adult cardiomyocytes (CMs) have poor proliferative capability, impairing functional recovery after heart injuries such as myocardial infarctions (MI), due to the inability to sufficiently replace damaged tissue. We have previously demonstrated in vivo that cardiomyocyte specific deletion of dual specificity tyrosine phosphorylation regulated kinase 1A (DYRK1A) induces an increase in CM cell cycle activity compared to wild type mice post-MI. Therefore, we hypothesized that pharmacological inhibition of DYRK1A would also promote CM cell cycle activity.

Methods: Neonatal rat cardiomyocytes (NRCMs) were isolated from 1–2-day old Sprague-Dawley pups. NRCMS were serum starved for 4 hours and treated with DYRK1A inhibitors LCTB-21 or LCTB-92 (0.01-1 µM) or respective isomeric and DMSO (0.01%) controls in serum-free media for 48 hours. NRCMS were fixed and stained for Ki67 (a cell cycling marker), cardiac troponin T, cyclin D1, and DAPI. Image analysis was performed using a CellProfiler pipeline to quantify the percentage of Ki67⁺ and cyclin D1⁺ CMs. Bulk RNA-seq from drug-treated and control NRCM conditions were examined using the DESeq2 package in R. In vivo, 12-week-old αDKRC::RLTG Ki67 reporter mice were induced with tamoxifen to label cycling CMs with GFP. MI I/R injuries were performed followed by oral gavage with LCTB-92 or iso-LCTB-92 for 7 days following injury and cycling CMs were quantified 4-weeks post-MI. Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparisons test and a Two-way ANOVA with Bonferroni correction for in vitro and in vivo data respectively.

Results/Conclusions: NRCM treatment with LCTB-21 (1 µM) and LCTB-92 (0.1 and 1 µM) significantly increased the percentage of Ki67⁺ and cyclin D1⁺ cells (p <0.05) compared to controls. Differentially expressed genes of LCTB-92 treated NRCMs were significantly enriched for GO terms related to cell cycle activity with respect to its isomeric control. In vivo, LCTB-92 treatment significantly increased the number of cycling CMs post-MI compared to controls (p <0.05). In conclusion, the pharmacological inhibition of DYRK1A induces cardiomyocyte cell cycle activity in vitro and in vivo, suggesting potential for clinical application in treating cardiac injury.