Isolation and functional characterization of alpha-smooth muscle actin expressing cardiomyocytes from embryonic stem cells.
Early mammalian heart development is characterized by transient expression of alpha-smooth muscle actin (Acta2). To date, cardiomyocytes expressing Acta2 in the early stages of in vivo development have not been characterized. To functionally characterize Acta2-expressing cardiomyocytes, we used a transgenic ES cell line expressing both the puromycin acetyl transferase (Pac) and enhanced green fluorescent protein (EGFP) cassettes under the control of the Acta2 promoter. The onset of Acta2 expression occurred in parallel with the appearance of beating areas, indicating the formation of cardiomyocytes. Antibiotic selection resulted in a high yield of cardiomyocytes and smooth muscle cells. The green fluorescent beating areas stained positively for multiple cardiomyocyte markers. Comparative electrophysiological analysis including fetal and alpha-MHC-expressing ES cell-derived cardiomyocyte controls showed that Acta2-positive cardiomyocytes contained pacemaker-, atrial- and ventricular-like phenotypes. Interestingly, the proportion of ventricular-like cells was much higher in the Acta2-positive cardiomyocytes population than in control alpha-MHC-expressing cardiomyocytes (75 % and 12 %, respectively). The findings of the present study provide a novel approach for the identification and enrichment of Acta2-positive cardiomyocytes, especially of the ventricular phenotype under in vitro conditions.
Center of Physiology and Pathophysiology, Institute of Neurophysiology, and Center of Molecular Medicine, University of Cologne (CMMC), Cologne, Germany.
Shiva Prasad
SP
Potta
Huamin
H
Liang
Johannes
J
Winkler
Michael Xavier
MX
Doss
Shuhua
S
Chen
Vilas
V
Wagh
Kurt
K
Pfannkuche
Jürgen
J
Hescheler
Agapios
A
Sachinidis
D000123:Acetyltransferases / Q000235:genetics; D000199:Actins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D000818:Animals; D002460:Cell Line; D002469:Cell Separation; D053595:Embryonic Stem Cells / Q000166:cytology*; D015870:Gene Expression; D049452:Green Fluorescent Proteins / Q000235:genetics; D051379:Mice; D009130:Muscle, Smooth / Q000378:metabolism*; D032383:Myocytes, Cardiac / Q000166:cytology / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019076:Transgenes
D000199:Actins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D000123:Acetyltransferases; C045336:puromycin N-acetyltransferase