Creating prodynorphin-expressing stem cells alerted for a high-throughput of cardiogenic commitment.
BACKGROUND
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.


RESULTS
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.


CONCLUSIONS
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.
Maioli
Margherita
M
Department of Biomedical Sciences and National Institute of Biostructures and Biosystems, University of Sassari, Sassari, Italy.
Asara
Yolande
Y
Pintus
Antonella
A
Ninniri
Stefania
S
Bettuzzi
Saverio
S
Scaltriti
Maurizio
M
Galimi
Francesco
F
Ventura
Carlo
C
D000818:Animals; D002454:Cell Differentiation / Q000502:physiology*; D002460:Cell Line; D003001:Cloning, Molecular; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000378:metabolism; D004745:Enkephalins / Q000378:metabolism*; D050991:GATA4 Transcription Factor / Q000378:metabolism; D000072576:Homeobox Protein Nkx-2.5; D018398:Homeodomain Proteins / Q000378:metabolism; D051379:Mice; D009200:Myocardial Contraction / Q000502:physiology; D009206:Myocardium / Q000378:metabolism*; D032383:Myocytes, Cardiac / Q000166:cytology* / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism; D011498:Protein Precursors / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism
D004745:Enkephalins; D050991:GATA4 Transcription Factor; C484171:Gata4 protein, mouse; D000072576:Homeobox Protein Nkx-2.5; D018398:Homeodomain Proteins; C484469:Nkx2-5 protein, mouse; D011498:Protein Precursors; D014157:Transcription Factors; C033660:preproenkephalin; D011493:Protein Kinase C