Effect of Calstabin1 Depletion on Calcium Transients and Energy Utilization in Muscle Fibers and Treatment Opportunities with RyR1 Stabilizers

Depletion of calstabin1 (FKBP12) from the RyR1 channel and consequential calcium leakage from the sarcoplasmic reticulum (SR) is found in certain disease conditions such as dystrophy, aging or muscle overuse. Here, we first assessed the effect of calstabin1 depletion on resting Ca2+ levels and transients. We found that depletion of calstabin1 with the calstabin1-dissociation compound FK506 increased the release of calcium from the SR by 14 % during tetanic stimulation (50 Hz, 300 ms) and delayed cytosolic calcium removal. However, we did not find a significant increase in resting cytosolic Ca2+ levels. Therefore, we tested if increased SERCA activity could counterbalance calcium leakage. By measuring the energy utilization of muscle fibers with and without FK506 treatment, we observed that FK506-treatment increased oxygen consumption by 125% compared to baseline levels. Finally, we found that pretreatment of muscle fibers with the RyR1 stabilizer JTV-519 led to an almost complete normalization of calcium flux dynamics and energy utilization. We conclude that cytosolic calcium levels are mostly preserved in conditions with leaky RyR1 channels due to increased SERCA activity. Therefore, we suggest that RyR1 leakiness might lead to chronic metabolic stress, followed by cellular damage, and RyR1 stabilizers could potentially protect diseased muscle tissue.publishe

Energy utilization in FDB fibers.

<p>The graph shows the estimates for the relative baseline change ± SEM after different treatment conditions (pretreatment/injection). Changes, displayed in %, are statistically valuated by the corresponding p-value. The increased SERCA activity observed after FK506 injection can be incompletely normalized by pretreatment with JTV-519.</p

Temporal profile curves for calcium transients during single twitch activation (A) and tetanic stimulation (B).

<p>Each line corresponds to the average of all single fiber measurements that have been included in the statistical analysis. For improving the visibility of group differences, the time interval was adjusted accordingly. The red and black dashed lines indicate the drift of the baseline calcium during the tetanic stimulation. Treatment of fibers with 25 μM FK506 (red line) leads to an elevated inter-peak-baseline level compared to untreated control fibers (black line). </p

The graph shows the estimates for the AUC (mean±SEM) for the four groups.

<p>Changes, displayed in %, are statistically valuated by the corresponding p-value. n: number of fibers measured per group. Control (n=42), FK506 (n=42), JTV-519 (n=53) and JTV-519 + FK506 (n= 44).</p