A Mechanism for Statin-Induced Susceptibility to Myopathy

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SUMMARY
This study aimed to identify a mechanism for statin-induced myopathy that explains its prevalence and selectivity for skeletal muscle, and to understand its interaction with moderate exercise. Statin-associated adverse muscle symptoms reduce adherence to statin therapy; this limits the effectiveness of statins in reducing cardiovascular risk. The issue is further compounded by perceived interactions between statin treatment and exercise. This study examined muscles from individuals taking statins and rats treated with statins for 4 weeks. In skeletal muscle, statin treatment caused dissociation of the stabilizing protein FK506 binding protein (FKBP12) from the sarcoplasmic reticulum (SR) calcium (Ca 2þ ) release channel, the ryanodine receptor 1, which was associated with pro-apoptotic signaling and reactive nitrogen species/reactive oxygen species (RNS/ ROS)Àdependent spontaneous SR Ca 2þ release events (Ca 2þ sparks). Statin treatment had no effect on Ca 2þ spark frequency in cardiac myocytes. Despite potentially deleterious effects of statins on skeletal muscle, there was no impact on force production or SR Ca 2þ release in electrically stimulated muscle fibers. Statin-treated rats with access to a running wheel ran further than control rats; this exercise normalized FKBP12 binding to ryanodine receptor 1, preventing the increase in Ca 2þ sparks and pro-apoptotic signaling. Statin-mediated RNS/ ROSÀdependent destabilization of SR Ca 2þ handling has the potential to initiate skeletal (but not cardiac) S tatins are the most widely prescribed drug in the Western world. Their use is predicted to rise further due to recent reductions in the cardiovascular risk threshold for statin prescription across the globe (1,2). However, cardiovascular benefits of statins are restricted by adverse effects that limit adherence (3,4) and, in turn, increase cardiovascular events (5) and mortality (6). The most common side effects and main reason for discontinuation of therapy emerge from skeletal muscle (statin myopathy or statin-associated adverse muscle symptoms). Although no strict definition of statin myopathy has been universally adopted (7)(8)(9)(10), we use this term to encompass the full spectrum of the effects of statins on skeletal muscle. This includes mild to moderate muscle symptoms and/or signs (myalgia: muscle pain with stiffness and weakness), as well as more severe potentially life-threatening outcomes (myositis and/or rhabdomyolysis) that are associated with raised creatine kinase (8,11). Although physical activity counteracts metabolic and cardiovascular diseases that are prevalent in subjects prescribed statins, exercise has been reported to exacerbate statin myopathy (12)(13)(14)(15)(16)(17)(18)(19), which may further limit the benefits of statins in those at risk of cardiovascular disease.
Statins are inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase that limit the production of cholesterol, isoprenoids, and coenzyme Q. Despite extensive research, which has focused on calcium (Ca 2þ ) homeostasis and mitochondrial function (20)(21)(22)(23)(24)(25), a cohesive mechanism for statin-induced myopathy is lacking. Furthermore, an understanding of why myopathy is not experienced by everyone who takes statins and the reason for its selectivity for skeletal muscle has not been fully addressed.
Using human and rodent muscle, we investigated the mechanism for statin-induced myopathy and described its interaction with voluntary moderate exercise. We revealed a mechanism by which statin treatment can make skeletal muscles susceptible to myopathy-dissociation of the FK506 binding protein (FKBP12) from the sarcoplasmic reticulum (SR) Ca 2þ release channel, the ryanodine receptor 1 (RyR1), which is accompanied by numerous spontaneous Ca 2þ release events (i.e., Ca 2þ sparks) (26). Statin treatment had no effect on Ca 2þ sparks in cardiac muscle.

Statin Myopathy
A U G U S T 2 0 1 9 : 5 0 9 -2 3  (27). FDB is predominantly type IIa; the choice of this muscle was informed by the short length of the fibers that allows the isolation and study of intact cells. In some cases, fibers were permeabilized by 2min exposure to 0.005% (w/v) saponin (28). Rat cardiac myocytes were isolated from Langendorffperfused hearts by collagenase and protease digestion (29).

Statin Myopathy
A U G U S T 2 0 1 9 : 5 0 9 -2 3 noteworthy that a robust dissociation of FKBP12 from RyR1 could be detected, although muscle biopsies were obtained from a diverse patient group ( Table 1).
FKBP12 dissociation from RyR1 has been shown to increase spontaneous SR Ca 2þ leak, which, in turn, promotes protein degradation and programmed cell death (33). Therefore, we next studied whether the change in the RyR complex in muscles of statintreated subjects was accompanied by indexes of pro-apoptotic signaling. For this purpose, we measured the protein expression of the inactive procaspase-3 and its cleaved active product, the proapoptotic enzyme caspase-3. Statin treatment increased caspase-3 expression in both human and rat muscles ( Figures 1C and 1D). In rat muscle, we also measured the proportion of TUNEL positive nuclei, which is another marker for pro-apoptotic signaling, and observed a marked increase with statin treatment ( Figure 1E) (20,21,23). However, all previous work has been performed on permeabilized muscle fibers, in which the constitutive inhibition of RyR1 by magnesium (37) and the dihydropyridine receptor (38) is reduced, which may mask the effects of statins. Therefore, we evaluated the effect of statin treatment on the SR  in Ca 2þ spark frequency or duration between fibers from control and statin-treated rats ( Figure 3C).
L-NAME inhibits NO and superoxide production from NOS (48), which indicates a role for NO, superoxide, and/or peroxynitrite in the spark-mediated leak.
Statin treatment has been shown to increase ROS production in skeletal muscle (25). We showed that these ROS played a role in the SR Ca 2þ leak, in Ca 2þ spark frequency and duration between fibers from control and statin-treated rats ( Figures 3D and 3E).
Bidirectional Ca 2þ fluxes between SR and mitochondria affect both SR and mitochondrial function.
Mitochondria accumulate close to SR Ca 2þ release sites during postnatal skeletal muscle maturation, which facilitates mitochondrial Ca 2þ uptake and is associated with reduced susceptibility to Ca 2þ spark activation (49). Conversely, excessive mitochondrial Ca 2þ uptake may promote Ca 2þ sparks by enhancing ROS production from complexes I and III (50,51). In support of this latter mechanism, the difference in Ca 2þ spark frequency and duration between fibers from control and statin-treated rats was no longer present after inhibiting Ca 2þ entry

Statin Myopathy
A U G U S T 2 0 1 9 : 5 0 9 -2 3  (13,14) and myositis (12,15,16), and that statins limit training adaptations in skeletal muscle (57,58). Therefore, we gave statin-treated and control rats access to an in-cage running wheel, which resulted in a type of voluntary exercise similar to that recommended for human subjects prescribed statins.

CONSEQUENCES OF STATIN-INDUCED SR
Rats were acclimatized to the wheel for 4 days before statin treatment commenced. Unexpectedly, the daily running distance was greater for statin-treated rats than for control rats across the 4 weeks of the study ( Figure 5A). The larger daily running distance in the statin group was due to an increase in the number of bouts of activity ( Figure 5B), whereas the running bout duration ( Figure 5C) and running velocity ( Figure 5D) were similar in the 2 groups.
In sharp contrast to the situation in muscles of sedentary subjects (see Figure 1), binding of FKBP12 to RyR1 showed no significant difference between muscles of statin-treated and control rats after 4 weeks of exercise ( Figures 6A and 6B). Moreover, in the exercised state, statin treatment no longer caused a significant increase in caspase 3 expression ( Figures 6C and 6D). Intriguingly, in the exercised state, the frequency of SR Ca 2þ sparks was lower in muscle fibers of statin-treated rats than in control rats, which contributed to a smaller spark-mediated Ca 2þ leak in this group (Figures 6E to 6G).
A number of studies have linked statin-induced myopathy with impaired mitochondrial biogenesis.  (Figures 7A to 7C).
Together, these data show that statin treatment did not limit moderate physical activity or markers of training adaptation in skeletal muscle. Exercise reversed the statin-dependent SR Ca 2þ leak, which suggests a potentially beneficial effect.

DISCUSSION
The prevalence of statin-induced muscle symptoms varies between 7% and 29% in registries and
In skeletal muscle of statin-treated humans and rats, we showed FKBP12 dissociation from RyR1, which resulted in a ROS/RNSÀdependent Ca 2þ sparkmediated SR Ca 2þ leak. Such destabilization of RyR1 has been associated with muscle dysfunction in a variety of conditions, including heart failure, aging, and muscular dystrophy (33)(34)(35). Accordingly, we observed indexes of pro-apoptotic signaling in statintreated subjects. Nevertheless, in the rodent model,  Lotteau et al.

Statin Myopathy
handling play an important role in the adaptation to endurance training (67)(68)(69)(70), and a moderate SR Ca 2þ leak has been linked to increased fatigue resistance (71,72). We showed beneficial effects of voluntary running exercise in muscle of statin-treated rats.  Lotteau et al.