CONTRACTILE ACTIVITY MODULATES ACTIN SYNTHESIS AND TURNOVER IN CULTURED NEONATAL RAT-HEART CELLS

In this study, the role that active tension development plays in the f ormation and maintenance of cardiac myocyte myofibrillar structure and cellular shape was investigated. By use of the calcium channel blocke r verapamil, spontaneous contractile activity of neonatal rat heart my ocytes was inhibited for 24 to 96 hours. Confocal microscopy of rhodam ine phalloidin-stained cells revealed that, within 24 hours of contrac tile arrest, actin filaments of myofibrils were no longer aligned with one another at their I bands and Z lines. Cellular shape was also aff ected, with the cells developing a less stellate appearance while rema ining attached to the substrate as well as to one another. By 48 hours , actin fibrils were largely absent from these cells. The disappearanc e of actin was confirmed by measurements of actin synthesis and accumu lation rates and by pulse-chase biosynthetic labeling experiments. It was revealed that, although actin synthesis was significantly reduced in arrested myocytes, the rapid disappearance of total cellular actin was largely due to increased rates of actin degradation. Contractile a rrest produced by L-type calcium channel blockade with verapamil (or o ther calcium channel blockers) accelerated actin degradation to a grea ter extent than K+ depolarization. Chloroquine partially suppressed th e accelerated rate of actin degradation, indicating that lysosomal pro teolysis may be involved in actin degradative processing. Protein kina se C activation also partially inhibited the accelerated rate of actin degradation but did not restore actin filaments in arrested myocytes. The reformation of actin fibrils and their reassembly into striated m yofibrils occurred when contractile activity was restored by removal o f verapamil from the culture medium. The period of time required for m yocytes to reassemble actin filaments and to regain their elongated mo rphology was proportional to the period of time that the cells were in hibited from contracting. Data are presented to indicate that active t ension development by neonatal cardiac myocytes in culture is critical to the maintenance of filamentous actin structure via mechanisms invo lving actin assembly, disassembly, and degradation.