Creating Prodynorphin-Expressing Stem Cells Alerted for a High-Throughput of Cardiogenic Commitment

Abstract

The development of cell therapy for the rescue of damaged heart muscle is a major area of inquiry. Within this context, the establishment of a cardiogenic cell line may remarkably facilitate the molecular dissection of cardiac fate specification, a low-efficiency and still poorly understood process, paving the way for novel approaches in the use of stem cells for cardiac repair. We used GTR1 cells, a derivative of mouse R1 embryonic stem cells bearing the puromycin-resistance gene driven by the cardiomyocyte-specific alpha-myosin heavy chain promoter, affording a gene trapping selection of a virtually pure population of embryonic stem cell-derived cardiomyocytes. Third-generation lentiviral vectors were used to overexpress the prodynorphin gene, previously shown to orchestrate a dynorphinergic system acting as a major conductor of embryonic stem cell cardiogenesis. Lentiviral prodynorphin transduction remarkably enhanced the transcription of GATA-4 and Nkx-2.5, two cardiac lineage-promoting genes, resulting in a dramatic increase in the number of spontaneously beating cardiomyocytes. Transduced cells also exhibited a subcellular redistribution patterning of protein kinase C-beta, -delta and -epsilon, a major requirement in cardiac lineage commitment. This activation resulted from a sustained increase in the transcription of targeted protein kinase C genes. Prodynorphin transduction was selective in nature and failed to activate genes responsible for skeletal myogenesis or neuronal specification. The cell line developed in this study provides a powerful in vitro model of cardiomyogenesis that may help clarify the cascade of transcriptional activation and signaling networks that push multipotent cells to take on the identity of a cardiac myocyte.