Remodeling of ryanodine receptor complex causes “leaky” channels: A molecular mechanism for decreased exercise capacity

To achieve uniform exercise, mice were placed in a pool and induced to swim twice daily for 3 weeks. After exercise, RyR1 was immunoprecipitated from hind-limb muscle homogenates and immunoblotted for channel complex components. Repeated exercise in the mouse resulted in progressive PKA phosphorylation of RyR1 at Ser-2844 that saturated by 14 days of twice-daily swimming for 90 min (Fig. 1A), and there was a significant increase in S-nitrosylation of free cysteines on RyR1 (Fig. 1A). In addition, the RyR1 macromolecular complex underwent remodeling, characterized by depletion of calstabin1 and PDE4D3 from the channel by day 14 (Fig. 1 A and B). A similar pattern of biochemical changes was seen in the EDL, soleus, tibialis anterior, gastrocnemius, and vastus muscles (data not shown). Only high-intensity exercise resulted in significant channel remodeling (Fig. 1 C and D). RyR1 remodeling persisted after exercise and recovered only partially after 3 days of rest [supporting information (SI) Fig. 5A]. PKA hyperphosphorylation of RyR1 was not due to changes in the amount of PKA or PP1 bound to the RyR1 complex (SI Fig. 5 A and B). Isolated EDL muscle strength was depressed after weeks of exercise, although it was able to recover over several days of rest (SI Fig. 5C). No significant histological evidence of muscle damage was apparent at the light microscopic level in EDL from WT mice following this exercise protocol (SI Fig. 5D). Total calstabin1 levels in whole-muscle homogenate, measured by immunoblot, were not altered during exercise (SI Fig. 5E). Twenty-one days of exercise resulted in an increase in endothelial nitric oxide synthase (eNOS) in EDL muscle (SI Fig. 5F) consistent with previous reports (17), which is a potential cause of increased S-nitrosylation of RyR1. Thus, fatiguing exercise over weeks causes PKA hyperphosphorylation, and S-nitrosylation of RyR1 and remodeling of the RyR1 complex manifested by depletion of PDE4D3 and calstabin1 from the channel.

To assess whether remodeling of the RyR1 channel macromolecular complex observed in exercised mice occurs in humans, thigh muscle biopsies were obtained from trained athletes before and after near-maximum aerobic exercise (Fig. 1E) (18). Exercise resulted in PKA hyperphosphorylation of RyR1, RyR1 S-nitrosylation, and calstabin1 depletion (n = 12 exercise) compared with controls (n = 6) (Fig. 1F). Before exercise on day 3, PKA phosphorylation of RyR1 in the trained cyclists was at or near resting levels, and no significant calstabin1 depletion from the RyR1 complex was observed; however, PDE4D3 was partially depleted from the RyR1 (Fig. 1F). These data suggest that similar remodeling of the RyR1 channel complex observed in mice occurs in highly trained athletes subjected to intense exercise.

We observed a significant defect in the exercise capacity of both muscle-specific cal1^−/− mice and PDE4D^−/− mice (predisposed to leaky PKA hyperphosphorylated RyR1 channels) compared with WT littermate controls (Fig. 2A). There was no correlation between exercise capacity and body weight (SI Fig. 6). Twenty-four hours after a downhill exercise regimen (see Materials and Methods for details), creatine phosphokinase (CPK) was significantly elevated consistent with increased muscle damage in both the cal1^−/− and PDE4D^−/− mice compared with WT (Fig. 2B). Interestingly, after several weeks of daily exercise training, the exercise capacity of WT mice approached that of cal1^−/−, presumably because of the progressive depletion of calstabin1 from the RyR1 complex due to PKA hyperphosphorylation of RyR1 that occurs with repeated exercise in WT mice (e.g., Fig. 1). PDE4D^−/− mice exhibited a significant increase in calstabin1 depletion from RyR1 after mild exercise (Fig. 2D). Thus, muscle-specific calstabin1- and PDE4D-deficient mice exhibited impaired exercise capacity.

We tested the effect of a drug the prevents depletion of calstabin1 from the RyR1 complex on exercise capacity. Derivatives of JTV519, a 1,4-benzothiazepine, were screened to identify compounds that enhance the binding affinity of calstabin1 to PKA phosphorylated and/or S-nitrosylated RyR, are specific for RyR1 (e.g., had no significant activity against other ion channels including HERG and voltage-gated Ca²⁺ channels), and have favorable drug-like properties (e.g., orally available, well absorbed, and stable). One compound that met these criteria, S107 (for synthesis, see SI Scheme 1), was selected (see SI Table 1)

Age- and sex-matched WT mice were randomized for implantation of osmotic pumps containing either S107 or vehicle. Treatment with S107 at 2.5 μg/hr or vehicle was initiated 4 days before the beginning of a 3-week daily swimming protocol. Exercise capacity was assessed once a week using a level treadmill run to exhaustion during the nocturnal cycle of the mouse. Fig. 3A shows that calstabin1 rebinding because of S107 treatment had no acute effect on WT exercise performance, but during daily exercise over 3 weeks, the S107 treated WT mice were relatively protected against a decline in treadmill exercise capacity that occurred in vehicle-treated mice (running time in minutes on day 21: 77.7 ± 4.6 exercise plus S107, n = 13 vs. 64.7 ± 1.4 exercise plus vehicle, n = 13; P < 0.05 Wilcoxon rank-sum test).

EDL muscles from S107-treated mice showed increased force production at stimulation frequencies >80 Hz (Fig. 3B). Exercise resulted in PKA phosphorylation and calstabin1 depletion from immunoprecipitated RyR1. Calstabin1 depletion from RyR1 was reversed by S107 treatment (Fig. 3C). S107 did not significantly improve exercise capacity in muscle-specific calstabin1-deficient mice (data not shown). Taken together, these data suggest that reducing SR Ca²⁺ leak with a drug that inhibits calstabin1 depletion from RyR1 can protect against muscle damage, enhance muscle function, and improve exercise capacity.

Immediately after the last session of the 3-week swimming/running exercise protocol flexor digitorum brevis (FDB) muscle fibers were enzymatically dissociated and loaded with the Ca²⁺ indicator fluo-4. Individual muscle fibers were imaged by using a Zeiss LSM 5 Live confocal microscope during field stimulation at 1 Hz and during a fatiguing protocol consisting of repeated 300-ms-long 120-Hz tetani every 2 seconds for 200 seconds. Representative calcium ΔF/F0 traces during the fatiguing stimulation protocol are shown for a FDB fiber isolated from a vehicle- (Fig. 4A) and S107-treated mouse (Fig. 4B). FDB fibers from S107-treated mice exhibited a delayed decline in peak tetanic Ca²⁺ transients (Fig. 4C). Muscle fibers with slower kinetics of Ca²⁺ release and reuptake are less prone to fatigue. Therefore, we also assessed the kinetics of Ca²⁺ release and reuptake during single twitches at 1 Hz. The distribution of 50% reuptake times (τ) showed no significant differences between vehicle and S107 treatment (SI Fig. 7), indicating no shift in the Ca²⁺ reuptake kinetics of the FDB fibers. These data indicate that treatment with S107 improves Ca²⁺ handling in muscle fibers and reduces muscle fatigue.

To determine whether the biochemical changes in the RyR1 macromolecular complex identified during exercise result in changes in RyR1 channel activity, single-channel function of RyR1 in SR microsomes from the hind-limb muscle of sedentary mice (sed), mice repeatedly exercised and treated with vehicle (Ex + veh), and mice repeatedly exercised and treated with S107 (Ex + S107) were determined in planar lipid bilayers. RyR1 channels were continuously measured for at least 10 min at 90 nM [Ca²⁺]_cis (Fig. 4D). Channels from exercised mice treated with vehicle displayed significantly higher open probabilities compared with channels from sedentary mice (P < 0.01, t test, Ex + veh, n = 9 vs. sed, n = 9) (Fig. 4E) or compared with those treated with S107 (P < 0.005, t test, Ex + S107, n = 12 vs. Ex + veh, n = 9). Thus, RyR1 channels from exercised animals, exhibited “leaky” channel behavior (increased open probability) and channels from animals treated with S107 were not leaky.

Ca²⁺ released into the cytosol via leaky RyR1 channels may activate calpain, Ca²⁺-dependent neutral proteases (19, 20). After repeated exercise, EDL muscle exhibited elevated calpain activity compared with sedentary controls, whereas calpain activity was significantly reduced in S107-treated mice (Fig. 4F). Evidence of protection against muscle damage was further provided by measurement of plasma CPK activity levels that were elevated in the exercised mice, but reduced close to the levels observed in sedentary controls in the S107-treated mice (Fig. 4G).

Our data suggest that remodeling of the RyR1 macromolecular complex during exercise, consisting of PKA hyperphosphorylation at Ser-2844, RyR1 S-nitrosylation, PDE4D3 depletion, and calstabin1 depletion, likely plays a role in determining exercise capacity. Exercise promotes numerous positive effects, from improvement in cardiovascular performance to increased glucose uptake and normalization of fuel metabolism (21, 22). On the other hand, exhausting exercise, such as that performed by a marathon runner or a long-distance cyclist, results in significant muscle damage and can impair task performance for days or weeks (23–25), although the mechanisms underlying this impairment in exercise capacity are not understood.

We identified biochemical changes in the RyR1 macromolecular complex consistent with leaky RyR1/Ca²⁺ release channels. Both muscle-specific deficiency of calstabin1 (cal1^−/−) or PDE4D3 (PDE4D^−/−) resulted in exercise defects in mice linking the observed remodeling of the RyR1 complex, characterized by calstabin1 and PDE4D3 depletion from the RyR1 complex, to impaired exercise performance. The Ca²⁺ channel stabilizer S107, which preserves binding of calstabin1 to RyR1 during exercise, improved exercise capacity in WT but not in cal1^−/− mice, demonstrating that the drug's mechanism of action requires calstabin1.

We propose that SR Ca²⁺ leak via RyR1 channels could result in muscle damage during intense exercise by activating calpain. Indeed, calpain activation and CPK levels were elevated after exercise and were reduced significantly by treatment with S107, suggesting that correction of leaky RyR1 may protect against muscle damage during exercise (Fig. 4). Our data do not exclude the possibility that other Ca²⁺-dependent pathways such as caspases, contribute to the damage induced by leaky RyR1 channels.

In summary, during exercise, remodeling of the RyR1 macromolecular complex results in leaky channels (because of depletion of calstabin1 from the channel complex) that play a role in limiting exercise capacity. The same physiological mechanisms that impair exercise capacity during chronic exercise are likely beneficial during acute exercise (including PKA phosphorylation and S-nitrosylation of RyR1, both of which activate the channel and may increase ECC gain).

We thank Susan Hamilton (Baylor College of Medicine, Houston), for muscle-specific FKBP12 (calstabin1)-deficient mice and helpful discussions and Bi-Xing Chen and Chiso Nwokafor for technical assistance. This work was supported by a grant from the Defense Advanced Research Projects Agency and an American Heart Association Scientist Development grant (to S.E.L.). A.R.M. and S.R. are consultants for a start-up company, ARMGO Pharma, Inc., that is targeting RyR1 to improve exercise capacity.