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CALCIUM-DRIVEN TRANSCRIPTION OF CARDIAC SPECIFYING GENE PROGRAM IN LIVER STEM CELLS
Citation:
MULLER-BORER, B., W. CASCIO, G. ESCH, D. W. GRAFF, A. AGHAJANIAN, J. LEMASTERS, W. COLEMAN, J. GRISHAM, AND N. MALOUF. CALCIUM-DRIVEN TRANSCRIPTION OF CARDIAC SPECIFYING GENE PROGRAM IN LIVER STEM CELLS. Presented at American Heart Association Conference, Dallas, TX, November 13 - 16, 2005.
Description:
We have previously shown that a cloned liver stem cell line (WB F344) acquires a cardiac phenotype when seeded in a cardiac microenvironment in vivo and ex vivo. Here we investigated the mechanisms of this transdifferentiation in early (<72 hr) WB F344 cell, rat neonatal ventricular myocyte co-cultures. We hypothesized that a signal transmitted from the myocyte into the WB F344 cells, via gap junctions, trigger the transcription of a cardiac specifying gene program and eventually a cardiac phenotype observed in the WB F344 cells. We assessed functional gap junction coupling in WB F344 cells expressing connexin 43 (Cx43) in co-culture using fluorescence recovery after photobleaching (n=30). Line scan confocal images recorded intracellular calcium ([Ca2+]i) signals in WB F344 cells and [Ca2+]i transients in adjacent myocytes (n=20). Real-time PCR and immunofluorescence monitored the expression of cardiac transcription factors, co-factors and cardiac specific proteins in the WB F344 cells. We found that in early co-cultures: 1) Physical contact between the WB F344 cells and adjacent myocytes was essential for the WB F344 cells to acquire a cardiac phenotype where functional gap junctions, most likely composed of Cx43, developed between the WB F344 cells and adjacent myocytes. 2) De novo oscillating [Ca2+]i spikes synchronous with [Ca2+]i transients in adjacent myocytes were asymmetrically located in the periphery of WB F344 cells adjacent to the myocyte. The [Ca2+]i signals recorded in WB F344 cells were inhibited with 100mM carbenoxolone, a gap junction uncoupler, but not in adjacent myocytes. 3) The multifunctional calcium/calmodulin sensitive CAmKinase II became preferentially phosphorylated in the WB F344 cells that were adjacent to myocytes. 4) Cardiac specifying transcription factors and co-factors, Nkx2.5, Mef2c and myocardin became expressed de novo, in the WB F344 cells 24 hrs after they were co-cultured with the myocytes. Cardiac Troponin I and T were expressed 5 days later. Collectively our results suggest that calcium signals transmitted into the WB F344 cells from adjacent myocytes via functional shared Cx43 gap junctions transduce the expression of a cardiac specifying gene program in the undifferentiated WB F344 cells.