CONDITIONAL DIFFERENTIATION OF HEART-DERIVED AND SMOOTH-MUSCLE-DERIVED CELLS TRANSFORMED BY A TEMPERATURE-SENSITIVE MUTANT OF SV40 T-ANTIGEN

To create muscle cell lines that conditionally differentiate in vitro we introduced a temperature-sensitive SV40 T antigen by retroviral inf ection into rat aortic smooth muscle cells (SMCs) and neonatal heart-d erived cells. After G418 selection cell lines isolated were characteri zed at permissive (33 degrees C) and non-permissive (39 degrees C) tem peratures. [H-3]Thymidine uptake showed that progression through the c ell cycle is greatly reduced at 39 degrees C. Cytoskeletal proteins, s uch as actins and vimentin did not change significantly after temperat ure shift, while the number of desmin-positive SMCs significantly incr eased when cells were switched to 39 degrees C. Heart-derived muscle c ells showed sarcomeric myosin heavy chain reactivity only when grown a t 39 degrees C. After thrombin stimulation intracellular calcium in bo th cell types increased severalfold in 39 degrees C-cells but not in 3 3 degrees C-cells. Whole cell patch-clamp recordings of SMCs and heart -derived cells revealed a strong increase in nicardipine-sensitive Ca2 + current when cells were switched to 39 degrees C. Nicardipine-insens itive Ca2+ current also increased in both cell types at the non-permis sive temperature. Na+ current in SMCs was large at 33 degrees C and sm all or not detectable at 39 degrees C and absent in heart-derived cell s. Using a cDNA probe specific for the alpha(1) subunit of the dihydro pyridine-sensitive Ca2+ channel we demonstrate a temperature-sensitive expression of the dihydropyridine receptor mRNA in smooth muscle-deri ved cells but not in heart-derived H10 cells. Our results suggest that upon downregulation of SV40 T antigen these cells become quiescent an d exhibit a more differentiated phenotype. These cell lines may provid e a useful tool to investigate ion channel- and receptor signal transd uction, as well as cell cycle control in smooth and possibly cardiac m uscle cell differentiation.