AIMS: To investigate the effects of static magnetic fields (MFs) on cardiomyogenesis of mouse embryonic stem (ES) cell-derived embryoid bodies and Flk-1(+) cardiac progenitor cells and to assess the impact of cytosolic calcium [Ca(2+)]c and reactive oxygen species (ROS). METHODS AND RESULTS: Embryoid bodies and ES cell-derived Flk-1(+) cardiovascular progenitor cells were exposed to static MFs. The expression of cardiac genes was evaluated by RT-PCR; sarcomeric structures were assessed by immunohistochemistry; intracellular ROS and [Ca(2+)]c of ES cells were examined by H2DCF-DA- and fluo-4-based microfluorometry. Treatment of embryoid bodies with MFs dose-dependent increased the number of contracting foci and cardiac areas as well as mRNA expression of the cardiac genes MLC2a, MLC2v, α-MHC and β-MHC. In Flk-1(+) cells MFs (1 mT) elevated both [Ca(2+)]c and ROS, increased expression of the cardiogenic transcription factors Nkx-2.5 and GATA-4 as well as cardiac genes. This effect was due to Ca(2+) influx, since extracellular Ca(2+) chelation abrogated ROS production and MF-induced cardiomyogenesis. Furthermore absence of extracellular calcium impaired sarcomere structures. Neither the phospholipase C inhibitor U73122 nor thapsigargin inhibited MF-induced increase in [Ca(2+)]c excluding involvement of intracellular calcium stores. ROS were generated through NAD(P)H oxidase, since NOX-4 but not NOX-1 and NOX-2 mRNA was upregulated upon MF exposure. Ablation of NOX-4 by sh-RNA and treatment with the NAD(P)H oxidase inhibitor diphenylen iodonium (DPI) totally abolished MF-induced cardiomyogenesis. CONCLUSION: The ability of static MFs to enhance cardiomyocyte differentiation of ES cells allows high throughput generation of cardiomyocytes without pharmacological or genetic modification.
AIMS: To investigate the effects of static magnetic fields (MFs) on cardiomyogenesis of mouse embryonic stem (ES) cell-derived embryoid bodies and Flk-1(+) cardiac progenitor cells and to assess the impact of cytosolic calcium [Ca(2+)]c and reactive oxygen species (ROS). METHODS AND RESULTS: Embryoid bodies and ES cell-derived Flk-1(+) cardiovascular progenitor cells were exposed to static MFs. The expression of cardiac genes was evaluated by RT-PCR; sarcomeric structures were assessed by immunohistochemistry; intracellular ROS and [Ca(2+)]c of ES cells were examined by H2DCF-DA- and fluo-4-based microfluorometry. Treatment of embryoid bodies with MFs dose-dependent increased the number of contracting foci and cardiac areas as well as mRNA expression of the cardiac genes MLC2a, MLC2v, α-MHC and β-MHC. In Flk-1(+) cells MFs (1 mT) elevated both [Ca(2+)]c and ROS, increased expression of the cardiogenic transcription factors Nkx-2.5 and GATA-4 as well as cardiac genes. This effect was due to Ca(2+) influx, since extracellular Ca(2+) chelation abrogated ROS production and MF-induced cardiomyogenesis. Furthermore absence of extracellular calcium impaired sarcomere structures. Neither the phospholipase C inhibitor U73122 nor thapsigargin inhibited MF-induced increase in [Ca(2+)]c excluding involvement of intracellular calcium stores. ROS were generated through NAD(P)H oxidase, since NOX-4 but not NOX-1 and NOX-2 mRNA was upregulated upon MF exposure. Ablation of NOX-4 by sh-RNA and treatment with the NAD(P)H oxidase inhibitor diphenylen iodonium (DPI) totally abolished MF-induced cardiomyogenesis. CONCLUSION: The ability of static MFs to enhance cardiomyocyte differentiation of ES cells allows high throughput generation of cardiomyocytes without pharmacological or genetic modification.