Abstract 1121: Ca2+ Influx through T-type Ca2+ Channels Is Necessary for Differentiation of Cardiac Stem Cells into Functional Cardiac Myocytes
Recent reports have shown that the heart contains a population of resident cardiac stem cells (CSCs) with the capacity to form new cardiac myocytes. The present study explored the hypothesis that Ca2+ influx through T-type Ca2+channels (TTCC), whose function is not established in the heart, is necessary for the differentiation of CSC into cardiac myocytes.
METHODS: Isolated c-Kit+(negative for hematopoietic markers) CSCs from young (4 months) adult feline hearts were infected with a control-EGFP-adenovirus (EGFP) prior to co-culture upon an existing neonatal rat ventricular myocyte (NRVM) feeder layer. In some experiments cKit+ CSC were infected with a shRNA-RFP-adenovirus targeted against all three feline TTCC genes (α1G, H and I) (Ad-AntiT). Co-cultured cells were immuno-stained for c-kit, α-actinin, Ki67, and α1G, H and I TTCC 1 or 2 days after co-culture and analyzed with confocal microscopy. TTCC mRNA levels in CSCs were analyzed using real time PCR. Contraction and Ca2+ transients (CaT) were measured in newly formed CSC derived myocytes were compared to NRVMs.
RESULTS: EGFP-CSCs differentiated into beating cardiac myocytes within 2 days of co-culturing. Fusion of CSC with NRVM was ruled out by infecting NRVM with RFP and CSC with GFP. Many differentiating CSC-derived cells stained positive for α-actinin (contractile protein marker) and many of these were Ki67+(proliferation marker) (48.4 ± 3.3 %; n=8 and 21.5 ± 4.6 %; n=5 respectively). Ad-AntiT reduced CSC differentiation into myocytes and reduced proliferation (α-actinin and Ki67 positive cells: 1.4 ± 0.5 %; n=8, and 3.0 ± 0.6 %; n=9 respectively; p<0.05). Real time PCR did not detect TTCC in isolated c-Kit+CSCs. After exposure to FGF and EGF, both α1G and α1I isotypes were observed. In co-culture, every CSC-derived myocyte undergoing mitosis and cytokinesis stained positive for α1G and α1I (n=20). In CSC-derived new myocytes the TTCC antagonist Ni2+(50 μM) significantly reduced Ca2+ transients (29%, P<0.05) and contractions (40%, P<0.05) while NRVM were not affected (P<0.53).
CONCLUSION: Expression of TTCC is necessary for CSC to differentiate into new cardiac myocytes, for their proliferation and initially for their contractile function.