Caffeine-induced Ca(2+) signaling as an index of cardiac progenitor cells differentiation.
Cardiac progenitor cells (CPCs), migrating from heart tissue, in culture aggregate to form cardiospheres (CSs) in which replication and cardiogenic differentiation occur. However, the frequency of functional differentiation in CSs and the role of cell clustering in supporting it remain to be established. The aim of our study is to quantify differentiation of a muscle-type Ca(2+) release mechanism in CS-derived cells, correlate it with cardiac differentiation markers and test its dependency on CS formation. CPCs migrating from murine cardiac explants were studied prior and after CSs formation (Pre-CS and Post-CS). Inducibility of RyR- and IP3-R-mediated Ca(2+) transients in individual cells was tested by exposure to caffeine and ATP, respectively; expression of cardiac and non-cardiac lineage markers was assessed. Caffeine responsiveness was negligible in Pre-CS cells and increased by 7.5 fold in Post-CS cells (3.6 vs. 26.9%; p < 0.05), and was closely correlated with activation of the cardiac TnI gene promoter. ATP-induced responses, frequent in Pre-CS (86%), were slightly increased in Post-CS cells (94%; p < 0.05). Expression of cardiac-specific Ca(2+)-handling proteins (Cav1.2, NCX1, RyR2, SERCA2a) was either limited to the Post-CS stage, or markedly enhanced. CS beating was infrequent, but its pharmacology was compatible with cardiac excitation-contraction coupling. Expression of non-cardiac lineage was low in general, and similar between Pre- and Post-CS cells. Culture conditions inhibiting CSs formation prevented the increase in caffeine responders. In conclusion, clustering in CSs leads to the induction of a muscle-specific functional response in about 30% of CPCs; this is accompanied by development of a cardiac-specific expression pattern.
Altomare
C
C
Department of Biotechnologies and Biosciences, University of Milano-Bicocca, p.za della Scienza 2, 20126, Milan, Italy.
Barile
L
L
Marangoni
S
S
Rocchetti
M
M
Alemanni
M
M
Mostacciuolo
G
G
Giacomello
A
A
Messina
E
E
Zaza
Antonio
A
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D002110:Caffeine / Q000494:pharmacology*; D020013:Calcium Signaling / Q000187:drug effects*; D002454:Cell Differentiation / Q000187:drug effects* / Q000235:genetics; D002465:Cell Movement; D002478:Cells, Cultured; D004558:Electric Stimulation; D005260:Female; D005786:Gene Expression Regulation; D053496:Inositol 1,4,5-Trisphosphate Receptors / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D024510:Muscle Development / Q000187:drug effects* / Q000235:genetics; D009200:Myocardial Contraction; D032383:Myocytes, Cardiac / Q000187:drug effects* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D019837:Ryanodine Receptor Calcium Release Channel / Q000378:metabolism; D013234:Stem Cells / Q000187:drug effects* / Q000378:metabolism; D013997:Time Factors; D019210:Troponin I / Q000235:genetics
D015415:Biomarkers; D053496:Inositol 1,4,5-Trisphosphate Receptors; D019837:Ryanodine Receptor Calcium Release Channel; D019210:Troponin I; D002110:Caffeine; D000255:Adenosine Triphosphate