Question: 

We have recently shown that TRPC6, a member of the transient receptor potential family of cation channels, is expressed in mouse skeletal muscle and is localized in the sarcolemma (Krüger et al. 2008, Neuromuscul Disord 18:501). Hyperforin, an activator of TRPC6 caused increases of intracellular Ca²⁺ in muscle fibers that could be partially blocked by ML-9. ML-9, known as a myosin light chain kinase inhibitor, is also a potent blocker of TRPC6, but does not affect other TRP channels. The current study was designed to investigate the functional role of TRPC6 for Ca²⁺ influx, muscle force and fatigue.

Methodology: 

To investigate divalent cation influx we used single interosseus muscle fibers and applied the Mn2+ quench technique using Fura-2. Quench of Fura-2 fluorescence was measured in response to excitation at 360 nm in the presence of 0.5 mM Mg²⁺. Muscle force of soleus muscles was recorded upon single stimuli (twitches) and repetitive stimuli at 10, 50 and 120 Hz. A fatigue protocol consisted of 50 Hz tetanic stimulation periods lasting 500 ms, interrupted by 1.5 s pauses.

Result: 

Background Ca²⁺ entry, as tested with the Mn²⁺ quench technique, was not affected by ML-9. However, application of 150 mM OAG, a TRPC6 activator stimulated background Ca²⁺ entry by about 50% (n=65 cells). Twitches, tetanic force at 50 Hz and 120 Hz stimulation were not affected by application of 50 mM ML-9 within 20 min. However, during sustained repetitive stimulation (fatigue protocol) force drop to half maximal force occurred 40% faster in the presence of ML-9 compared to control (n=6/10 muscles tested). Even after a recovery period tetanic force was reduced by 25 % in the presence of ML-9. Therefore we hypothesized that TRPC6 might play a role in the recovery from fatigue. Thus, we studied the kinetics of force increase (recovery) after a 260 s lasting fatigue protocol leading to force drop to about 30% of the initial value. When 50 mM ML-9 was applied after the fatigue protocol, complete recovery was observed within 20 min, both in the control and in the ML-9 group (n = 9/9 muscles tested).

Conclusion: 

We conclude that TRPC6 is present in the sarcolemma of mouse skeletal muscle and can be pharmacologically activated. TRPC6 does not seem to have a significant open probability at rest. Further, we have no indication that TRPC6 is activated during recovery from fatigue, e.g. by stimulating Ca²⁺ influx from extracellular. The fact that ML-9 accelerates force drop during the fatigue protocol indicates that TRPC6 is activated during sustained repetitive muscle stimulation counteracting fatigue. However, it can not be excluded that ML-9 modulates muscle force via alternative intracellular mechanisms.