Effect of Triadin on Retrograde and Orthograde Signaling between RyR1 and DHPR in Cultured Myotubes

Abstract

Using pan triadin-null mice we previously showed that triadins ablation did not disrupt EC-coupling in muscle cells. However, calcium imaging studies in cultured myotubes did reveal that triadin-null myotubes had slightly smaller depolarization-induced Ca2+ transients than Wt cells. Here, using whole-cell voltage clamp, we analyze the effect of triadin ablation in skeletal EC-coupling by characterizing the retrograde and orthograde signaling between RyR1 and DHPR of triadin-null myotubes. Calcium currents elicited by 200ms depolarization steps in Wt and triadin-null cells showed slow kinetics of activation and peak current at approximately +30 mV. Although, the overall voltage dependence was preserved between Wt and triadin-null cells a leftward shift in the I/V curve was observed in triadin-null cells (V1/2, 22.3±0.8 mV in Wt vs 16.6±1.1 mV in triadin-null cells, p<0.05). In addition, kinetic analysis of the DHPR Ca2+ current shows that the activation time constant of the slow component (τslow) was slightly decreased from 37±2.4 ms in Wt to 26±2.6 ms (p<0.05) in triadin-null cells.The voltage-evoked Ca2+ transient, on the other hand, showed a small but significant reduction of the peak fluorescence amplitude of triadin-null cells (ΔF /Fmax, 0.72±0.2 in Wt vs 0.61±0.1 in triadin-null) with no differences in voltage dependence (Vm, −7.2±1.1 mV in Wt vs −10.1±1.9 mV in null cells). Our results suggest that the absence of triadin expression preserves the orthograde and retrograde signaling between DHPR and RyR1 nearly intact and that the effect of triadin ablation on ΔF/Fmax would be secondary to the dysregulation of calcium homeostasis observed in triadin-null cells. These data give further support to the idea that skeletal triadins do not play a direct role in skeletal EC-coupling.Supported by NIH Grants 5K01AR054818-02 (to CFP) and 1P01AR044750 (to PDA).