Effect of the Non-Immunosuppressive MPT Pore Inhibitor Alisporivir on the Functioning of Heart Mitochondria in Dystrophin-Deficient mdx Mice
Abstract
:1. Introduction
2. Materials and Methods
2.1. Animals
2.2. ECG
2.3. Electron Microscopy
2.4. Mitochondria Isolation and Determination of Respiration and Oxidative Phosphorylation
2.5. Determination of Ca2+ Retention by Mitochondria, MPT Pore Opening Assay
2.6. Lipid Peroxidation
2.7. RNA Extraction, Reverse Transcription, and Quantitative Real-Time PCR
2.8. Quantification of Mitochondrial DNA
2.9. Electrophoresis and Immunoblotting of Mitochondrial OXPHOS Proteins
2.10. Statistical Analysis
3. Results
3.1. Effect of Alisporivir on the Intensity of the Inflammatory Process in mdx Mice
3.2. Effect of Alisporivir on the Ultrastructure of Heart Mitochondria
3.3. Impact of Alisporivir on the Functioning of Heart Mitochondria
3.4. Effect of Alisporivir on the Expression of Proteins Responsible for Mitochondrial Biogenesis and Mitochondrial Dynamics
3.5. Impact of Alisporivir on Some Parameters of the Heart Muscle
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
- Emery, A.E. Population frequencies of inherited neuromuscular diseases—A world survey. Neuromuscul. Disord. 1991, 1, 19–29. [Google Scholar] [CrossRef]
- Romitti, P.A.; Zhu, Y.; Puzhankara, S.; James, K.A.; Nabukera, S.K.; Zamba, G.K.; Ciafaloni, E.; Cunniff, C.; Druschel, C.M.; Mathews, K.D.; et al. Prevalence of Duchenne and Beckermuscular dystrophies in the United States. Pediatrics 2015, 135, 513–521. [Google Scholar] [CrossRef] [Green Version]
- Ignatieva, E.; Smolina, N.; Kostareva, A.; Dmitrieva, R. Skeletal Muscle Mitochondria Dysfunction in Genetic Neuromuscular Disorders with Cardiac Phenotype. Int. J. Mol. Sci. 2021, 22, 7349. [Google Scholar] [CrossRef] [PubMed]
- Verhaert, D.; Richards, K.; Rafael-Fortney, J.A.; Raman, S.V. Cardiac involvement in patients with muscular dystrophies magnetic resonance imaging phenotype and genotypic considerations. Circ. Cardiovasc. Imaging 2011, 4, 67–76. [Google Scholar] [CrossRef] [Green Version]
- Hughes, M.C.; Ramos, S.V.; Turnbull, P.C.; Rebalka, I.A.; Cao, A.; Monaco, C.M.F.; Varah, N.E.; Edgett, B.A.; Huber, J.S.; Tadi, P.; et al. Early myopathy in Duchenne muscular dystrophy is associated with elevated mitochondrial H2O2 emission during impaired oxidative phosphorylation. J. Cachexia Sarcopenia Muscle 2019, 10, 643–661. [Google Scholar] [CrossRef] [Green Version]
- Dubinin, M.V.; Talanov, E.Y.; Tenkov, K.S.; Starinets, V.S.; Mikheeva, I.B.; Sharapov, M.G.; Belosludtsev, K.N. Duchenne muscular dystrophy is associated with the inhibition of calcium uniport in mitochondria and an increased sensitivity of the organelles to the calcium-induced permeability transition. Biochim. Biophys. Acta Mol. Basis Dis. 2020, 1866, 165674. [Google Scholar] [CrossRef] [PubMed]
- Dubinin, M.V.; Talanov, E.Y.; Tenkov, K.S.; Starinets, V.S.; Belosludtseva, N.V.; Belosludtsev, K.N. The Effect of Deflazacort Treatment on the Functioning of Skeletal Muscle Mitochondria in Duchenne Muscular Dystrophy. Int. J. Mol. Sci. 2020, 21, 8763. [Google Scholar] [CrossRef]
- Bodensteiner, J.B.; Engel, A.G. Intracellular calcium accumulation in Duchenne dystrophy and other myopathies: A study of 567,000 muscle fibers in 114 biopsies. Neurology 1978, 28, 439–446. [Google Scholar] [CrossRef] [PubMed]
- Millay, D.P.; Sargent, M.A.; Osinska, H.; Baines, C.P.; Barton, E.R.; Vuagniaux, G.; Sweeney, H.L.; Robbins, J.; Molkentin, J.D. Genetic and pharmacologic inhibition of mitochondrial-dependent necrosis attenuates muscular dystrophy. Nat. Med. 2008, 14, 442–447. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Reutenauer, J.; Dorchies, O.M.; Patthey-Vuadens, O.; Vuagniaux, G.; Ruegg, U.T. Investigation of Debio 025, a cyclophilin inhibitor, in the dystrophic mdx mouse, a model for Duchenne muscular dystrophy. Br. J. Pharmacol. 2008, 155, 574–584. [Google Scholar] [CrossRef] [Green Version]
- Wissing, E.R.; Millay, D.P.; Vuagniaux, G.; Molkentin, J.D. Debio-025 is more effective than prednisone in reducing muscular pathology in mdx mice. Neuromuscul. Disord 2010, 20, 753–760. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Schiavone, M.; Zulian, A.; Menazza, S.; Petronilli, V.; Argenton, F.; Merlini, L.; Sabatelli, P.; Bernardi, P. Alisporivir rescues defective mitochondrial respiration in Duchenne muscular dystrophy. Pharmacol. Res. 2017, 125, 122–131. [Google Scholar] [CrossRef] [PubMed]
- Hansson, M.J.; Mattiasson, G.; Mansson, R.; Karlsson, J.O.; Keep, M.F.; Ruegg, U.T.; Dumont, J.-M.; Besseghir, K.; Elmér, E. The nonimmunosuppressive cyclosporin analogs NIM811 and UNIL025 display nanomolar potencies on permeability transition in brain-derived mitochondria. J. Bioenerg. Biomembr. 2004, 36, 407–413. [Google Scholar] [CrossRef]
- Stupka, N.; Gregorevic, P.; Plant, D.R.; Lynch, G.S. The calcineurin signal transduction pathway is essential for successful muscle regeneration in mdx dystrophic mice. Acta Neuropathol. 2004, 107, 299–310. [Google Scholar] [CrossRef]
- Ascah, A.; Khairallah, M.; Daussin, F.; Bourcier-Lucas, C.; Godin, R.; Allen, B.G.; Petrof, B.J.; Rosiers, C.D.; Burelle, Y. Stress-induced opening of the permeability transition pore in the dystrophin-deficient heart is attenuated by acute treatment with sildenafil. Am. J. Physiol. Heart Circ. Physiol. 2011, 300, H144–H153. [Google Scholar] [CrossRef] [PubMed]
- Dubinin, M.V.; Talanov, E.Y.; Tenkov, K.S.; Starinets, V.S.; Mikheeva, I.B.; Belosludtsev, K.N. Transport of Ca2+ and Ca2+-dependent permeability transition in heart mitochondria in the early stages of Duchenne muscular dystrophy. Biochim. Biophys. Acta Bioenerg. 2020, 1861, 148250. [Google Scholar] [CrossRef] [PubMed]
- Angebault, C.; Panel, M.; Lacôte, M.; Rieusset, J.; Lacampagne, A.; Fauconnier, J. Metformin Reverses the Enhanced Myocardial SR/ER-Mitochondria Interaction and Impaired Complex I-Driven Respiration in Dystrophin-Deficient Mice. Front. Cell Dev. Biol. 2021, 8, 609493. [Google Scholar] [CrossRef] [PubMed]
- Astashev, M.E.; Serov, D.A.; Tankanag, A.V. Anesthesia effects on the low frequency blood flow oscillations in mouse skin. Skin Res. Technol. 2019, 25, 40–46. [Google Scholar] [CrossRef] [PubMed]
- Kawai, S.; Takagi, Y.; Kaneko, S.; Kurosawa, T. Effect of three types of mixed anesthetic agents alternate to ketamine in mice. Exp. Anim. 2011, 60, 481–487. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Tsukamoto, A.; Serizawa, K.; Sato, R.; Yamazaki, J.; Inomata, T. Vital signs monitoring during injectable and inhalant anesthesia in mice. Exp. Anim. 2015, 64, 57–64. [Google Scholar] [CrossRef] [Green Version]
- Shin, J.H.; Nitahara-Kasahara, Y.; Hayashita-Kinoh, H.; Ohshima-Hosoyama, S.; Kinoshita, K.; Chiyo, T.; Okada, H.; Okada, T.; Takeda, S. Improvement of cardiac fibrosis in dystrophic mice by rAAV9-mediated microdystrophin transduction. Gene Ther. 2011, 18, 910–919. [Google Scholar] [CrossRef]
- Belosludtsev, K.N.; Belosludtseva, N.V.; Kosareva, E.A.; Talanov, E.Y.; Gudkov, S.V.; Dubinin, M.V. Itaconic acid impairs the mitochondrial function by the inhibition of complexes II and IV and induction of the permeability transition pore opening in rat liver mitochondria. Biochimie 2020, 176, 150–157. [Google Scholar] [CrossRef]
- Belosludtseva, N.V.; Starinets, V.S.; Pavlik, L.L.; Mikheeva, I.B.; Dubinin, M.V.; Belosludtsev, K.N. The Effect of S-15176 Difumarate Salt on Ultrastructure and Functions of Liver Mitochondria of C57BL/6 Mice with Streptozotocin/High-Fat Diet-Induced Type 2 Diabetes. Biology 2020, 9, 309. [Google Scholar] [CrossRef]
- Ye, J.; Coulouris, G.; Zaretskaya, I.; Cutcutache, I.; Rozen, S.; Madden, T. Primer-BLAST: A tool to design target-specific primers for polymerase chain reaction. BMC Bioinform. 2012, 13, 134. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Schmittgen, T.D.; Livak, K.J. Analyzing real-time PCR data by the comparative C(T) method. Nat. Protoc. 2008, 3, 1101–1108. [Google Scholar] [CrossRef]
- Quiros, P.M.; Goyal, A.; Jha, P.; Auwerx, J. Analysis of mtDNA/nDNA ratio in mice. Curr. Protoc. Mouse Biol. 2017, 7, 47–54. [Google Scholar] [CrossRef] [Green Version]
- Pant, M.; Sopariwala, D.H.; Bal, N.C.; Lowe, J.; Delfín, D.A.; Rafael-Fortney, J.; Periasamy, M. Metabolic dysfunction and altered mitochondrial dynamics in the utrophin-dystrophin deficient mouse model of Duchenne muscular dystrophy. PLoS ONE 2015, 10, e0123875. [Google Scholar] [CrossRef] [Green Version]
- De Mario, A.; Gherardi, G.; Rizzuto, R.; Mammucari, C. Skeletal muscle mitochondria in health and disease. Cell Calcium 2021, 94, 102357. [Google Scholar] [CrossRef]
- Statile, C.J.; Taylor, M.D.; Mazur, W.; Cripe, L.H.; King, E.; Pratt, J.; Benson, D.W.; Hor, K.N. Left ventricular noncompaction in Duchenne muscular dystrophy. J Cardiovasc. Magn. Reson. 2013, 15, 67. [Google Scholar] [CrossRef] [Green Version]
- Stocco, A.; Smolina, N.; Sabatelli, P.; Šileikytė, J.; Artusi, E.; Mouly, V.; Cohen, M.; Forte, M.; Schiavone, M.; Bernardi, P. Treatment with a triazole inhibitor of the mitochondrial permeability transition pore fully corrects the pathology of sapje zebrafish lacking dystrophin. Pharmacol. Res. 2021, 165, 105421. [Google Scholar] [CrossRef] [PubMed]
- Porter, G.A., Jr.; Beutner, G. Cyclophilin D, Somehow a Master Regulator of Mitochondrial Function. Biomolecules 2018, 8, 176. [Google Scholar] [CrossRef] [Green Version]
- Amanakis, G.; Murphy, E. Cyclophilin D: An Integrator of Mitochondrial Function. Front. Physiol. 2020, 11, 595. [Google Scholar] [CrossRef]
- Kirschner, J.; Schessl, J.; Schara, U.; Reitter, B.; Stettner, G.M.; Hobbiebrunken, E.; Wilichowski, E.; Bernert, G.; Weiss, S.; Stehling, F.; et al. Treatment of Duchenne muscular dystrophy with ciclosporin A: A randomised, double-blind, placebo-controlled multicentre trial. Lancet Neurol. 2010, 9, 1053–1059. [Google Scholar] [CrossRef]
- Stuckey, D.J.; Carr, C.A.; Camelliti, P.; Tyler, D.J.; Davies, K.E.; Clarke, K. In vivo MRI characterization of progressive cardiac dysfunction in the mdx mouse model of muscular dystrophy. PLoS ONE 2012, 7, e28569. [Google Scholar] [CrossRef]
- Boyman, L.; Chikando, A.C.; Williams, G.S.; Khairallah, R.J.; Kettlewell, S.; Ward, C.W.; Smith, G.L.; Kao, J.P.; Lederer, W.J. Calcium movement in cardiac mitochondria. Biophys. J. 2014, 107, 1289–1301. [Google Scholar] [CrossRef] [Green Version]
- Williams, G.S.; Boyman, L.; Chikando, A.C.; Khairallah, R.J.; Lederer, W.J. Mitochondrial calcium uptake. Proc. Natl. Acad. Sci. USA 2013, 110, 10479–10486. [Google Scholar] [CrossRef] [Green Version]
- Voit, A.; Patel, V.; Pachon, R.; Shah, V.; Bakhutma, M.; Kohlbrenner, E.; McArdle, J.J.; Dell’Italia, L.J.; Mendell, J.R.; Xie, L.H.; et al. Reducing sarcolipin expression mitigates Duchenne muscular dystrophy and associated cardiomyopathy in mice. Nat. Commun. 2017, 8, 1068. [Google Scholar] [CrossRef] [Green Version]
- Rybalka, E.; Timpani, C.A.; Cooke, M.B.; Williams, A.D.; Hayes, A. Defects in mitochondrial ATP synthesis in dystrophin deficient Mdx skeletal muscles may be caused by complex I insufficiency. PLoS ONE 2014, 9, e115763. [Google Scholar] [CrossRef] [Green Version]
- Winter, J.; Hammer, E.; Heger, J.; Schultheiss, H.P.; Rauch, U.; Landmesser, U.; Dörner, A. Adenine Nucleotide Translocase 1 Expression is Coupled to the HSP27-Mediated TLR4 Signaling in Cardiomyocytes. Cells 2019, 8, 1588. [Google Scholar] [CrossRef] [Green Version]
- Yapa, N.M.B.; Lisnyak, V.; Reljic, B.; Ryan, M.T. Mitochondrial dynamics in health and disease. FEBS Lett. 2021, 595, 1184–1204. [Google Scholar] [CrossRef]
- Oka, S.I.; Sabry, A.D.; Cawley, K.M.; Warren, J.S. Multiple Levels of PGC-1α Dysregulation in Heart Failure. Front Cardiovasc. Med. 2020, 7, 2. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Belosludtseva, N.V.; Starinets, V.S.; Mikheeva, I.B.; Serov, D.A.; Astashev, M.E.; Belosludtsev, M.N.; Dubinin, M.V.; Belosludtsev, K.N. Effect of the MPT pore inhibitor alisporivir on the development of mitochondrial dysfunction in the heart tissue of diabetic mice. Biology 2021, 10, 839. [Google Scholar] [CrossRef]
- Qi, R.; Wang, D.; Xing, L.; Wu, Z. Cyclosporin A inhibits mitochondrial biogenesis in Hep G2 cells. Biochem. Biophys. Res. Commun. 2018, 496, 941–946. [Google Scholar] [CrossRef] [PubMed]
- Elrod, J.W.; Wong, R.; Mishra, S.; Vagnozzi, R.J.; Sakthievel, B.; Goonasekera, S.A.; Karch, J.; Gabel, S.; Farber, J.; Force, T.; et al. Cyclophilin D controls mitochondrial pore-dependent Ca(2+) exchange, metabolic flexibility, and propensity for heart failure in mice. J. Clin. Investig. 2010, 120, 3680–3687. [Google Scholar] [CrossRef] [PubMed]
Gene | Forward (5′ → 3′) | Reverse (5′ → 3′) |
---|---|---|
Ant1 | CTATGACACTGCCAAGGGGATG | TCAAACGGATAGGACACCAGC |
Ant2 | TCTGGACGCAAAGGAACTGA | GACCATGCGCCCTTGAAA |
Ppif | GCAGATGTCGTGCCAAAGACTG | GCCATTGTGGTTGGTGAAGTCG |
Drp1 | TTACAGCACACAGGAATTGT | TTGTCACGGGCAACCTTTTA |
Mfn2 | CACGCTGATGCAGACGGAGAA | ATCCCAGCGGTTGTTCAGG |
Ppargc1a | CTGCCATTGTTAAGACCGAG | GTGTGAGGAGGGTCATCGTT |
Rplp2 | CGGCTCAACAAGGTCATCAGTGA | AGCAGAAACAGCCACAGCCCCAC |
Nd4 | ATTATTATTACCCGATGAGGGAACC | ATTAAGATGAGGGCAATTAGCAGT |
Gapdh | GTGAGGGAGATGCYCAGTGT | CTGGCATTGCTCTCAATGAC |
Groups | Creatine Kinase | LDH | AST |
---|---|---|---|
U/L | |||
WT (n = 10) | 349.7 ± 74.4 | 349.5 ± 43.0 | 62.3 ± 9.9 |
WT + Ali (n = 9) | 460.5 ± 79.8 | 260.0 ± 36.1 | 40.4 ± 7.6 |
mdx (n = 8) | 3376.7 ± 670.6 *# | 1355.4 ± 264.7 *# | 414.0 ± 63.2 *# |
mdx + Ali (n = 10) | 2103.8 ± 316.7 *# | 950.9 ± 106.1 *# | 252.3 ± 23.5 *#¥ |
Animal (n = 5) | State 2 | State 3 | State 4 | State 3UDNP | RC | ADP/O |
---|---|---|---|---|---|---|
nmol O2/min per 1 mg of Protein | Relative Units | |||||
WT | 11.4 ± 3.0 | 37.6 ± 2.9 | 14.6 ± 1.5 | 38.3 ± 3.3 | 2.6 ± 0.2 | 2.2 ± 0.1 |
WT + Ali | 17.1 ± 1.6 | 52.5 ± 4.3 | 15.3 ± 1.0 | 50.1 ± 3.8 | 3.4 ± 0.2 | 2.5 ± 0.2 |
mdx | 14.3 ± 0.9 | 60.5 ± 7.3 * | 17.4 ± 2.0 | 63.7 ± 7.6 * | 3.5 ± 0.2 * | 2.5 ± 0.1 |
mdx + Ali | 15.4 ± 1.2 | 53.7 ± 4.1 | 15.7 ± 1.3 | 53.5 ± 4.0 | 3.4 ± 0.2 | 2.3 ± 0.1 |
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. |
© 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
Share and Cite
Dubinin, M.V.; Starinets, V.S.; Talanov, E.Y.; Mikheeva, I.B.; Belosludtseva, N.V.; Serov, D.A.; Tenkov, K.S.; Belosludtseva, E.V.; Belosludtsev, K.N. Effect of the Non-Immunosuppressive MPT Pore Inhibitor Alisporivir on the Functioning of Heart Mitochondria in Dystrophin-Deficient mdx Mice. Biomedicines 2021, 9, 1232. https://doi.org/10.3390/biomedicines9091232
Dubinin MV, Starinets VS, Talanov EY, Mikheeva IB, Belosludtseva NV, Serov DA, Tenkov KS, Belosludtseva EV, Belosludtsev KN. Effect of the Non-Immunosuppressive MPT Pore Inhibitor Alisporivir on the Functioning of Heart Mitochondria in Dystrophin-Deficient mdx Mice. Biomedicines. 2021; 9(9):1232. https://doi.org/10.3390/biomedicines9091232
Chicago/Turabian StyleDubinin, Mikhail V., Vlada S. Starinets, Eugeny Yu. Talanov, Irina B. Mikheeva, Natalia V. Belosludtseva, Dmitriy A. Serov, Kirill S. Tenkov, Evgeniya V. Belosludtseva, and Konstantin N. Belosludtsev. 2021. "Effect of the Non-Immunosuppressive MPT Pore Inhibitor Alisporivir on the Functioning of Heart Mitochondria in Dystrophin-Deficient mdx Mice" Biomedicines 9, no. 9: 1232. https://doi.org/10.3390/biomedicines9091232