Effect of Exercise on Statin-Induced Skeletal Muscle Myopathy Senior Thesis Alay S. Parikh The School of Molecular and Cellular Biology University of Illinois at Urbana-Champaign Research Advisor: Associate Professor Marni D. Boppart, ScD. Department of Kinesiology and Community Health University of Illinois at Urbana-Champaign
24
Embed
Effect of Exercise on Statin-induced Myopathy (Parikh)
This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Transcript
Effect of Exercise on Statin-Induced Skeletal Muscle Myopathy
Senior Thesis
Alay S. Parikh
The School of Molecular and Cellular Biology
University of Illinois at Urbana-Champaign
Research Advisor:
Associate Professor Marni D. Boppart, ScD.
Department of Kinesiology and Community Health
University of Illinois at Urbana-Champaign
1
Abstract
HMG-CoA reductase inhibitors, or statins, significantly decrease
hypercholesterolemia and protect against cardiovascular disease. Statins directly inhibit
the action of the HMG-CoA reductase enzyme in the cholesterol synthesis pathway. One
of the most common side effects of statin treatment is skeletal muscle myopathy, a
condition that may be exacerbated by exercise. Our recent results suggest that short-
term exercise does not aggravate statin-induced myopathy as assessed by force
production, but may initiate myofiber damage and atrophy in a manner that can result in
exacerbated myopathy with long-term statin administration. The purpose of this study was
to directly assess myofiber damage and atrophy in hypercholesterolemic mice (ApoE-/-)
treated with statins and exercise. Male, ApoE-/- mice were provided access to a running
wheel for two weeks, then continued exercise for an additional two weeks while receiving
daily injections of simvastatin or saline treatment (accustomed exercise). A second
exercise group received access to a running week for two weeks concomitant with
simvastatin or saline treatment (no prior training, novel exercise). A control group received
simvastatin or saline treatment for two weeks without access to a running wheel
(sedentary). There was no evidence of overt fiber damage as a result of statin
administration in the absence or presence of exercise. However, Type 2B fiber size was
significantly reduced in the accustomed exercise group that received statin therapy. The
results from this study suggest that statin treatment may stimulate early myofiber
degradation of Type 2 fibers when combined with an exercise training program.
2
Introduction
Cardiovascular disease (CVD) is the leading cause of death for both men and
women in the United States. One of the most common and effective preventative
treatments to combat CVD is statin therapy. Statins work by inhibiting HMG-CoA
reductase, the rate-limiting enzyme in the cholesterol biogenesis pathway. High
cholesterol levels in the blood (hypercholesterolemia) increase the risk of CVD via an
increase in atherosclerosis within the circulatory system. As such, statins are one of the
most widely prescribed pharmaceutical agents administered to prevent the development
of CVD. However, a relatively common side effect of statin usage is the occurrence of
skeletal muscle myopathy (Sinzinger 2002; Ucar 2012). The mechanism behind the
relationship between statins and myopathy remains poorly understood, and elucidating
the underlying molecular mechanisms is of prime importance to reduce the incidence of
statin-induced myopathy.
Different mechanisms have been proposed to explain the mechanistic basis for
statin-induced myopathy. The primary theory supported by literature is an increase in
mitochondrial dysfunction corresponding with a depletion of Coenzyme Q10 in the muscle
(Duncan 2008, Vaklavas 2009). This mitochondrial co-factor serves as an electron carrier
within the electron transport chain. Reductions in Coenzyme Q10 can result in an
abnormal accumulation of reactive oxygen species (ROS) within the mitochondria, which
can ultimately induce cellular apoptosis, DNA damage, and enzyme inactivation (Slimen
2014). Coenzyme Q10 is generated downstream of mevalonate (a major product in the
early stages of cholesterol biogenesis). Thus, statin usage inhibits the tissue’s normal
ability to synthesize Coenzyme Q10, which can result in enhanced mitochondrial
3
dysfunction and altered cellular respiration. This dysfunction could be responsible for the
skeletal muscle myopathy and myalgia experienced by statin users.
Cholesterol is vital to the stability and structure of the skeletal muscle membrane,
or sarcolemma. Statin-induced disruptions to cholesterol synthesis may significantly
impair muscle membrane flexibility, allowing for damage as a result of contraction and
mechanical strain (Parker 2012). Thus, a reduction in cholesterol represents an
alternative explanation for statin-induced myopathy.
A main preventative measure recommended for individuals at risk for CVD is to
undertake an aerobic exercise training program (American Heart Association 2013).
Aerobic exercise training has repeatedly been demonstrated to improve cardiovascular
function as well as reduce high blood pressure and the risk for CVD (Gielen 2015).
Despite the fact that exercise is an important component of the prescription for CVD,
studies suggest that exercise in combination with statin therapy may exacerbate
myopathy. Parker et al. (2012) and others have demonstrated that exercise in
combination with statin therapy can increase the prevalence of muscle pain and reduce
engagement in physical activity. Therefore, further studies are necessary to provide a
better understanding of the biological basis for this condition.
Our lab recently conducted a study that attempted to address the mechanisms
that underlie statin-induced myopathy and the impact of exercise on this condition
(Boppart lab, unpublished results, manuscript in progress). Hypercholesterolemic mice
(ApoE-/-) received either statin therapy or saline then were subjected to statin treatment
after two weeks of running wheel activity (accustomed exercise group, total of 4 weeks of
exercise), statin treatment concurrent with exposure to running wheel activity (novel
4
exercise group, 2 weeks of exercise), or remained sedentary throughout the treatment.
Statin treatment resulted in significant myopathy as assessed by total running wheel
activity, hindlimb grip strength, and maximal isometric force. Exercise, either accustomed
or novel, did not provoke further deficits in activity or force. However, gene expression of
an important ubiquitin ligase, atrogin-1, was significantly elevated in the exercise groups
in combination with statin administration. Systemic inflammation, as assessed by serum
amyloid A, was also significantly elevated in the novel exercise group in combination with
statins. Therefore, these results suggest the ability for exercise to stimulate the
degradation of myofibrillar protein during statin administration.
The purpose of this study was to extend the results of our primary investigation
and determine the extent to which exercise can increase myofiber damage and atrophy
in the presence of statin therapy. We hypothesized, based on previous results, that
exercise would provoke a significant increase in both myofiber damage and atrophy, and
that these early events may contribute to exacerbation of myopathy observed with long-
term statin administration.
Methods
Study Design. 8 week-old male ApoE knockout mice (ApoE-/-) (Jackson Laboratory, Bar
Harbor, ME) were randomized to six groups (n=10/group). Mice were first assigned to
one of three groups: no exercise, voluntary novel exercise (initiation of exercise at 2
weeks, concomitant to initiation of statin or placebo), or voluntary accustomed exercise
(exercise starting 2 weeks prior to statin or placebo administration and during treatment).
Exercise was administered through the use of a running wheel. Mice were injected with
5
either simvastatin (20 mg/kg/day) or an equivalent volume saline. Groups are designated
as Sedentary+Saline (n=3), Sedentary+Statin (n=3-4), Novel+Saline (n=3), Novel+Statin
(n=3-4), Accustomed+Saline (n=3), and Accustomed+Statin (n=2). Due to the original
samples being used for the initial study, the number of samples available for this study
was limited.
Immunofluorescence. Gastrocnemius-soleus muscle complexes previously frozen in
isopentane were divided at the midline along the axial plane, and the distal half was
embedded in OCT (Tissue-Tek; Fischer Scientific). Three transverse cryosections per
sample (10μm non-serial sections, each separated by a minimum of 40 μm) were cut for
each histological assessment using a CM3050S cryostat (Lecia, Wezlar, Germany).
Sections were placed on microscope slides (Superfrost; Fischer Scientific, Hanover Park,
IL) and stored at -80°C before staining.
To assess skeletal muscle damage, sections were stained with anti-IgG antibodies
and dystrophin to distinguish individual muscle fibers. The frozen tissue sections (10μm)
were fixed in ice cold acetone and blocked with AffiniPure Fab Fragments Goat Anti-
Figure 1. The effect of statin treatment and exercise on myofiber damage and regeneration. (A) The ratio of IgG+ myofibers per area of muscle (mm2) was assessed to determine the amount of myofiber damage that occurred during a combination of statin and exercise therapy. (B) Percentage of myofibers displaying a centrally-located nucleus, a hallmark of damage and repair/regeneration. No significant differences were detected between groups. Figure 2. The effect of exercise and statin administration on mean cross sectional area. (A) The mean myofiber CSA, as well as the mean CSA for (B) Type 2A, (C) Type 2X, and (D) Type 2B fibers were assessed. No significant differences were detected between groups. Figure 3. Fiber type-specific size distribution is differentially affected by a combination of exercise and statin therapy. The percentage of (A) Type 2A, (B) Type 2X, and (C) Type 2B myofibers categorized by size. The combination of statin and exercise altered Type 2X and Type 2B myofiber size. *p<0.05 compared to all other
groups; ǂ†*p<0.05 compared to Sedentary+Statin, Novel+Saline, Accustomed+Saline; ǂ
*p<0.05 compared to Sedentary+Saline, Accustomed+Saline. Figure 4. The percentage of Type 2 fibers is selectively affected by a combination of exercise and statin therapy. The percentage of (A) Type 2A, (B) Type 2X, and (C) Type 2B fibers was unaffected by statin or exercise alone; however, a trend for a statin x exercise interaction effect was observed for Type 2X (p=0.07) and Type 2B (p=0.06).
20
Acknowledgements
I would like to thank Michael Munroe for his guidance and support throughout the entire
project. He has been extremely helpful as a mentor and an important factor in the
completion of my senior thesis. I would also like to thank my faculty advisor, Dr. Marni
Boppart, for her support and providing me with the opportunity to complete this work. I
would also like to acknowledge Dr. Hae R. Chung for completing the initial study and
performing all the functional tests with mice. I would also like to thank Ziad Mahmassani
and Slav Dvoretskiy for their assistance with this project. This work was supported by a
grant from the Center for Health Aging and Disease (to MDB).
21
References
1. Sinzinger, H. W., Roswitha‡; Peskar, Bernhard A.§ (2002). "Muscular Side Effects of Statins." Journal of Cardiovascular Pharmacology 40(2): 163-171. 2. Ucar, M., T. Mjörndal and R. Dahlqvist (2012). "HMG-CoA Reductase Inhibitors and Myotoxicity." Drug Safety 22(6): 441-457 3. Larsen, S., N. Stride, M. Hey-Mogensen, C. N. Hansen, L. E. Bang, H. Bundgaard, L. B. Nielsen, J. W. Helge and F. Dela (2013). "Simvastatin Effects on Skeletal MuscleRelation to Decreased Mitochondrial Function and Glucose 4. Parker, B. A. and P. D. Thompson (2012). "Effect of Statins on Skeletal Muscle: Exercise, Myopathy, and Muscle Outcomes." Exercise and sport sciences reviews 40(4): 188-194. 5. Bruckert, E., G. Hayem, S. Dejager, C. Yau and B. Begaud (2005). "Mild to moderate muscular symptoms with high-dosage statin therapy in hyperlipidemic patients--the PRIMO study." Cardiovasc Drugs Ther 19(6): 403-414. Intolerance." Journal of the American College of Cardiology 61(1): 44-53. 6. Menshikova, E. V., V. B. Ritov, L. Fairfull, R. E. Ferrell, D. E. Kelley and B. H. Goodpaster (2006). "Effects of Exercise on Mitochondrial Content and Function in Aging Human Skeletal Muscle." J Gerontol A Biol Sci Med Sci 61(6): 534-540. 7. Meador, B. M. and K. A. Huey (2011). "Statin-associated changes in skeletal muscle function and stress response after novel or accustomed exercise." Muscle & Nerve 44(6): 882-889. 8. Smit, J. W. A., P. R. BÄR, R. A. Geerdink† and D. W. Erkelens (1995). "Heterozygous familial hypercholesterolaemia is associated with pathological exercise-induced leakage of muscle proteins, which is not aggravated by simvastatin therapy." European Journal of Clinical Investigation 25(2): 79-84. 9. Schoenfeld, B. J. (2012). "Does exercise-induced muscle damage play a role in skeletal muscle hypertrophy?" J Strength Cond Res 26(5): 1441-1453.
10. Hirst, J., Martin S. King and Kenneth R. Pryde (2008). "The production of reactive oxygen species by complex I." Biochemical Society Transactions 36(5): 976-980. 11. Kang, J., H. Albadawi, V. I. Patel, T. A. Abbruzzese, J. H. Yoo, W. G. Austen, Jr. and M. T. Watkins (2008). "Apolipoprotein E-/- mice have delayed skeletal muscle healing after hind limb ischemia-reperfusion." J Vasc Surg 48(3): 701-708.
22
12. Murlasits, Z. and Z. Radák (2014). "The Effects of Statin Medications on Aerobic Exercise Capacity and Training Adaptations." Sports Medicine 44(11): 1519-1530. 13. Rahimov, F. and L. M. Kunkel (2013). "Cellular and molecular mechanisms underlying muscular dystrophy." The Journal of Cell Biology 201(4): 499-510. 14. Rockl, K. S., M. F. Hirshman, J. Brandauer, N. Fujii, L. A. Witters and L. J. Goodyear (2007). "Skeletal muscle adaptation to exercise training: AMP-activated protein kinase mediates muscle fiber type shift." Diabetes 56(8): 2062-2069. 15. Wernig, A., A. Irintchev and P. Weisshaupt (1990). "Muscle injury, cross-sectional area and fibre type distribution in mouse soleus after intermittent wheel-running." J Physiol 428: 639-652.
16. Costill, D. L., E. F. Coyle, W. F. Fink, G. R. Lesmes and F. A. Witzmann (1979). "Adaptations in skeletal muscle following strength training." Journal of Applied Physiology 46(1): 96-99. 17. Dirks, A. J. and K. M. Jones (2006). "Statin-induced apoptosis and skeletal myopathy." American Journal of Physiology - Cell Physiology 291(6): C1208-C1212. 18. Murlasits, Z. and Z. Radák (2014). "The Effects of Statin Medications on Aerobic Exercise Capacity and Training Adaptations." Sports Medicine 44(11): 1519-1530. 19. Phillips, P. S., R. H. Haas, S. Bannykh, S. Hathaway, N. L. Gray, B. J. Kimura, G. D. Vladutiu and J. D. F. England (2002). "Statin-Associated Myopathy with Normal Creatine Kinase Levels." Annals of Internal Medicine 137(7): 581.
20. Maxwell, A. J., J. Niebauer, P. S. Lin, P. S. Tsao, D. Bernstein and J. P. Cooke (2009). "Hypercholesterolemia impairs exercise capacity in mice." Vascular Medicine 14(3): 249-257. 21. Thompson PD, Zmuda JM, Domalik LJ, Zimet RJ, Staggers J and Guyton JR (1997). Lovastatin increases exercise-induced skeletal muscle injury. Metabolism 46: 1206-10.
22. Bouitbir J, Charles AL, Rasseneur L, Dufour S, Piquard F, Geny B and Zoll J (2011). Atorvastatin treatment reduces exercise capacities in rats: involvement of mitochondrial impairments and oxidative stress. J Appl Physiol 111: 1477-83.
23. Mikus CR, Boyle LJ, Borengasser SJ, Oberlin DJ, Naples SP, Fletcher J, Meers GM, Ruebel M, Laughlin MH, Dellsperger KC, Fadel PJ and Thyfault JP (2013). Simvastatin impairs exercise training adaptations. J Am Coll Cardiol 62: 709-14.
24. Tomlinson SS and Mangione KK (2005). Potential adverse effects of statins on muscle. Phys Ther 85: 459-65.
23
25. Calvacanti, G. M.; Oliveira, A. S. B.; Assis, T. O.; Chimelli, L. M. C.; Madeiros, P. L. & Mota, D. L. (2011). Histochemistry and morphometric analysis of muscle fibers from patients with Duchenne muscular dystrophy (DMD). Int. J. Morphol., 29(3):934-938.
26. Olsen, D. B., A. R. Langkilde, M. C. Orngreen, E. Rostrup, M. Schwartz and J. Vissing (2003). "Muscle structural changes in mitochondrial myopathy relate to genotype." J Neurol 250(11): 1328-1334.
27. Ahn, S. C. (2008). "Neuromuscular complications of statins." Phys Med Rehabil Clin N Am 19(1): 47-59, vi.
28. Warren, G. L., D. A. Hayes, D. A. Lowe and R. B. Armstrong (1993). "Mechanical factors in the initiation of eccentric contraction-induced injury in rat soleus muscle." J Physiol 464: 457-475.
29. Andrew J. Duncan, Iain P. Hargreaves, Maxwell S. Damian, John M. Land & Simon J. R. Heales (2009) Decreased Ubiquinone Availability and Impaired Mitochondrial Cytochrome Oxidase Activity Associated With Statin Treatment, Methods, 19:1, 44-50, DOI: 10.1080/15376510802305047
30. Gielen, S., M. H. Laughlin, C. O’Conner and D. J. Duncker (2014) "Exercise Training in Patients with Heart Disease: Review of Beneficial Effects and Clinical Recommendations." Progress in Cardiovascular Diseases 57(4): 347-355.
31. Slimen, I. B., T. Najar, A. Ghram, H. Dabbebi, M. Ben Mrad and M. Abdrabbah (2014). "Reactive oxygen species, heat stress and oxidative-induced mitochondrial damage. A review." Int J Hyperthermia 30(7): 513-523.
32. Vaklavas, C., Y. S. Chatzizisis, A. Ziakas, C. Zamboulis and G. D. Giannoglou (2009). "Molecular basis of statin-associated myopathy." Atherosclerosis 202(1): 18-28.
33. Eckel RH, Jakicic JM, Ard, JD, Hubbard VS, de Jesus JM, Lee IM, Lichtenstein AH, Loria CM, Millen BE, Houston Miller N, Nonas CA, Sacks FM, Smith SC Jr, Svetkey LP, Wadden TW, Yanovski SZ. (2013) AHA/ACC guideline on lifestyle management to reduce cardiovascular risk: a report of the Cardiology American/Heart Association Task Force on Practice Guidelines. Circulation. 2013;00:000–000.