Iron limitation promotes the atrophy of skeletal myocytes, whereas iron supplementation prevents this process in the hypoxic conditions

Kobak,Kamil; Kasztura,Monika; Dziegala,Magdalena; Bania,Jacek; Kapuśniak,Violetta; Banasiak,Waldemar; Ponikowski,Piotr; Jankowska,Ewa  A.

doi:10.3892/ijmm.2018.3481

Iron limitation promotes the atrophy of skeletal myocytes, whereas iron supplementation prevents this process in the hypoxic conditions

Authors:
- Kamil Kobak
- Monika Kasztura
- Magdalena Dziegala
- Jacek Bania
- Violetta Kapuśniak
- Waldemar Banasiak
- Piotr Ponikowski
- Ewa A. Jankowska
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Affiliations: Laboratory for Applied Research on Cardiovascular System, Department of Heart Diseases, Wrocław Medical University, 50‑981 Wrocław, Poland, Department of Food Hygiene and Consumer Health, Wroclaw University of Environmental and Life Sciences, 50‑375 Wroclaw, Poland, Department of Histology and Embryology, Wroclaw University of Environmental and Life Sciences, 50‑375 Wroclaw, Poland, Centre for Heart Diseases, Military Hospital, 50‑981 Wrocław, Poland
Published online on: February 12, 2018 https://doi.org/10.3892/ijmm.2018.3481
Pages: 2678-2686
Copyright: © Kobak et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

There is clinical evidence that patients with heart failure and concomitant iron deficiency have increased skeletal muscle fatigability and impaired exercise tolerance. It was expected that a skeletal muscle cell line subjected to different degrees of iron availability and/or concomitant hypoxia would demonstrate changes in cell morphology and in the expression of atrophy markers. L6G8C5 rat skeletal myocytes were cultured in normoxia or hypoxia at optimal, reduced or increased iron concentrations. Experiments were performed to evaluate the iron content in cells, cell morphology, and the expression of muscle specific atrophy markers [Atrogin1 and muscle‑specific RING‑finger 1 (MuRF1)], a gene associated with the atrophy/hypertrophy balance [mothers against decapentaplegic homolog 4 (SMAD4)] and a muscle class‑III intermediate filament protein (Desmin) at the mRNA and protein level. Hypoxic treatment caused, as compared to normoxic conditions, an increase in the expression of Atrogin‑1 (P<0.001). Iron‑deficient cells exhibited morphological abnormalities and demonstrated a significant increase in the expression of Atrogin‑1 (P<0.05) and MuRF1 (P<0.05) both in normoxia and hypoxia, which indicated activation of the ubiquitin proteasome pathway associated with protein degradation during muscle atrophy. Depleted iron in cell culture combined with hypoxia also induced a decrease in SMAD4 expression (P<0.001) suggesting modifications leading to atrophy. In contrast, cells cultured in a medium enriched with iron during hypoxia exhibited inverse changes in the expression of atrophy markers (both P<0.05). Desmin was upregulated in cells subjected to both iron depletion and iron excess in normoxia and hypoxia (all P<0.05), but the greatest augmentation of mRNA expression occurred when iron depletion was combined with hypoxia. Notably, in hypoxia, an increased expression of Atrogin‑1 and MuRF1 was associated with an increased expression of transferrin receptor 1, reflecting intracellular iron demand (R=0.76, P<0.01; R=0.86, P<0.01). Hypoxia and iron deficiency when combined exhibited the most detrimental impact on skeletal myocytes, especially in the context of muscle atrophy markers. Conversely, iron supplementation in in vitro conditions acted in a protective manner on these cells.

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1	Cohen S, Nathan JA and Goldberg AL: Muscle wasting in disease: Molecular mechanisms and promising therapies. Nat Rev Drug Discov. 14:58–74. 2015. View Article : Google Scholar : PubMed/NCBI
2	Jackman RW and Kandarian SC: The molecular basis of skeletal muscle atrophy. Am J Physiol Cell Physiol. 287:C834–C843. 2004. View Article : Google Scholar : PubMed/NCBI
3	Sillau AH and Banchero N: Effects of hypoxia on capillary density and fiber composition in rat skeletal muscle. Pflugers Arch. 370:227–232. 1977. View Article : Google Scholar : PubMed/NCBI
4	Zattara-Hartmann MC, Badier M, Guillot C, Tomei C and Jammes Y: Maximal force and endurance to fatigue of respiratory and skeletal muscles in chronic hypoxemic patients: The effects of oxygen breathing. Muscle Nerve. 18:495–502. 1995. View Article : Google Scholar : PubMed/NCBI
5	Sanders KJ, Kneppers AE, van de Bool C, Langen RC and Schols AM: Cachexia in chronic obstructive pulmonary disease: New insights and therapeutic perspective. J Cachexia Sarcopenia Muscle. 7:5–22. 2016. View Article : Google Scholar : PubMed/NCBI
6	Clark AL, Poole-Wilson PA and Coats AJ: Exercise limitation in chronic heart failure: Central role of the periphery. J Am Coll Cardiol. 28:1092–1102. 1996. View Article : Google Scholar : PubMed/NCBI
7	Coats A: The 'Muscle Hypothesis' of chronic heart failure. J Mol Cell Cardiol. 28:2255–2262. 1996. View Article : Google Scholar : PubMed/NCBI
8	Coats AJ, Clark AL, Piepoli M, Volterrani M and Poole-Wilson PA: Symptoms and quality of life in heart failure: The muscle hypothesis. Br Heart J. 72(suppl 2): S36–S39. 1994. View Article : Google Scholar : PubMed/NCBI
9	Piepoli MF, Kaczmarek A, Francis DP, Davies LC, Rauchhaus M, Jankowska EA, Anker SD, Capucci A, Banasiak W and Ponikowski P: Reduced peripheral skeletal muscle mass and abnormal reflex physiology in chronic heart failure. Circulation. 114:126–134. 2006. View Article : Google Scholar : PubMed/NCBI
10	Mancini DM, Walter G, Reichek N, Lenkinski R, McCully KK, Mullen JL and Wilson JR: Contribution of skeletal muscle atrophy to exercise intolerance and altered muscle metabolism in heart failure. Circulation. 85:1364–1373. 1992. View Article : Google Scholar : PubMed/NCBI
11	Farkas J, von Haehling S, Kalantar-Zadeh K, Morley JE, Anker SD and Lainscak M: Cachexia as a major public health problem: Frequent, costly, and deadly. J Cachexia Sarcopenia Muscle. 4:173–178. 2013. View Article : Google Scholar : PubMed/NCBI
12	Hajahmadi M, Shemshadi S, Khalilipur E, Amin A, Taghavi S, Maleki M, Malek H and Naderi N: Muscle wasting in young patients with dilated cardiomyopathy. J Cachexia Sarcopenia Muscle. 8:542–548. 2017. View Article : Google Scholar : PubMed/NCBI
13	Buller NP, Jones D and Poole-Wilson PA: Direct measurement of skeletal muscle fatigue in patients with chronic heart failure. Br Heart J. 65:20–24. 1991. View Article : Google Scholar : PubMed/NCBI
14	Stugiewicz M, Tkaczyszyn M, Kasztura M, Banasiak W, Ponikowski P and Jankowska EA: The influence of iron deficiency on the functioning of skeletal muscles: Experimental evidence and clinical implications. Eur J Heart Fail. 18:762–773. 2016. View Article : Google Scholar : PubMed/NCBI
15	Loncar G, Springer J, Anker M, Doehner W and Lainscak M: Cardiac cachexia: Hic et nunc. J Cachexia Sarcopenia Muscle. 7:246–260. 2016. View Article : Google Scholar : PubMed/NCBI
16	Zizola C and Schulze PC: Metabolic and structural impairment of skeletal muscle in heart failure. Heart Fail Rev. 18:623–630. 2013. View Article : Google Scholar :
17	Gosker HR, Wouters EF, van der Vusse GJ and Schols AM: Skeletal muscle dysfunction in chronic obstructive pulmonary disease and chronic heart failure: Underlying mechanisms and therapy perspectives. Am J Clin Nutr. 71:1033–1047. 2000. View Article : Google Scholar : PubMed/NCBI
18	von Haehling S, Lainscak M, Springer J and Anker SD: Cardiac cachexia: A systematic overview. Pharmacol Ther. 121:227–252. 2009. View Article : Google Scholar
19	Minotti JR, Christoph I, Oka R, Weiner MW, Wsells L and Massie BM: Impaired skeletal muscle function in patients with congestive heart failure. Relationship to systemic exercise performance. J Clin Invest. 88:2077–2082. 1991. View Article : Google Scholar : PubMed/NCBI
20	Jankowska EA, Rozentryt P, Witkowska A, Nowak J, Hartmann O, Ponikowska B, Borodulin-Nadzieja L, Banasiak W, Polonski L, Filippatos G, et al: Iron deficiency: An ominous sign in patients with systolic chronic heart failure. Eur Heart J. 31:1872–1880. 2010. View Article : Google Scholar : PubMed/NCBI
21	Jankowska EA, Malyszko J, Ardehali H, Koc-Zorawska E, Banasiak W, von Haehling S, Macdougall IC, Weiss G, McMurray JJ, Anker SD, et al: Iron status in patients with chronic heart failure. Eur Heart J. 34:827–834. 2013. View Article : Google Scholar :
22	Anker SD, Comin Colet J, Filippatos G, Willenheimer R, Dickstein K, Drexler H, Lüscher TF, Bart B, Banasiak W, Niegowska J, et al: Ferric carboxymaltose in patients with heart failure and iron deficiency. N Engl J Med. 361:2436–2448. 2009. View Article : Google Scholar : PubMed/NCBI
23	Okonko DO, Grzeslo A, Witkowski T, Mandal AKJ, Slater RM, Roughton M, Foldes G, Thum T, Majda J, Banasiak W, et al: Effect of intravenous iron sucrose on exercise tolerance in anemic and nonanemic patients with symptomatic chronic heart failure and iron deficiency. J Am Coll Cardiol. 51:103–112
24	Dziegala M, Kasztura M, Kobak K, Bania J, Banasiak W, Ponikowski P and Jankowska EA: Influence of the availability of iron during hypoxia on the genes associated with apoptotic activity and local iron metabolism in rat H9C2 cardiomyocytes and L6G8C5 skeletal myocytes. Mol Med Rep. 14:3969–3977. 2016. View Article : Google Scholar : PubMed/NCBI
25	Gomes MD, Lecker SH, Jagoe RT, Navon A and Goldberg AL: Atrogin-1, a muscle-specific F-box protein highly expressed during muscle atrophy. Proc Natl Acad Sci. 98:14440–14445. 2001. View Article : Google Scholar : PubMed/NCBI
26	Bodine SC, Latres E, Baumhueter S, Lai VK, Nunez L, Clarke BA, Poueymirou WT, Panaro FJ, Na E, Dharmarajan K, et al: Identification of ubiquitin ligases required for skeletal muscle atrophy. Science. 294:1704–1708. 2001. View Article : Google Scholar : PubMed/NCBI
27	Palus S, von Haehling S and Springer J: Muscle wasting: An overview of recent developments in basic research. J Cachexia Sarcopenia Muscle. 5:193–198. 2014. View Article : Google Scholar : PubMed/NCBI
28	Liu F, Pouponnot C and Massagué J: Dual role of the Smad4/DPC4 tumor suppressor in TGFbeta-inducible transcriptional complexes. Genes Dev. 11:3157–3167. 1997. View Article : Google Scholar
29	Seong HA, Jung H, Kim KT and Ha H: 3-Phosphoinositide-dependent PDK1 negatively regulates transforming growth factor-β-induced signaling in a kinase-dependent manner through physical interaction with Smad Proteins. J Biol Chem. 282:12272–12289. 2007. View Article : Google Scholar : PubMed/NCBI
30	Sartori R, Schirwis E, Blaauw B, Bortolanza S, Zhao J, Enzo E, Stantzou A, Mouisel E, Toniolo L, Ferry A, et al: BMP signaling controls muscle mass. Nat Genet. 45:1309–1318. 2013. View Article : Google Scholar : PubMed/NCBI
31	Aliparasti MR, Alipour MR, Almasi S and Feizi H: Ghrelin administration increases the Bax/Bcl-2 gene expression ratio in the heart of chronic hypoxic rats. Adv Pharm Bull. 5:195–199. 2015. View Article : Google Scholar : PubMed/NCBI
32	Fletcher J: Iron transport in the blood. Proc R Soc Med. 63:1216–1218. 1970.PubMed/NCBI
33	Woo KJ, Lee TJ, Park JW and Kwon TK: Desferrioxamine, an iron chelator, enhances HIF-1alpha accumulation via cyclooxy-genase-2 signaling pathway. Biochem Biophys Res Commun. 343:8–14. 2006. View Article : Google Scholar : PubMed/NCBI
34	Parkes JG, Hussain RA, Olivieri NF and Templeton DM: Effects of iron loading on uptake, speciation, and chelation of iron in cultured myocardial cells. J Lab Clin Med. 122:36–47. 1993.PubMed/NCBI
35	Hoepken HH, Korten T, Robinson SR and Dringen R: Iron accumulation, iron-mediated toxicity and altered levels of ferritin and transferrin receptor in cultured astrocytes during incubation with ferric ammonium citrate. J Neurochem. 88:1194–1202. 2004. View Article : Google Scholar : PubMed/NCBI
36	Chandio ZA, Talpur FN, Khan H, Afridi HI and Khaskheli GQ: Determination of cadmium and zinc in vegetables with online FAAS after simultaneous pre-concentration with 1,5-diphenylthiocarbazone immobilised on naphthalene. Food Addit Contam Part A Chem Anal Control Expo Risk Assess. 30:110–115. 2013. View Article : Google Scholar
37	Lowry OH, Rosebrough NJ, Farr AL and Randall RJ: Protein measurement with the folin phenol reagent. J Biol Chem. 193:265–275. 1951.PubMed/NCBI
38	Pfaffl MW: A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 29:e452001. View Article : Google Scholar : PubMed/NCBI
39	Walker JM: The bicinchoninic acid (BCA) assay for protein quantitation. Methods Mol Biol. 32:5–8. 1994.PubMed/NCBI
40	Ikeda Y, Imao M, Satoh A, Watanabe H, Hamano H, Horinouchi Y, Izawa-Ishizawa Y, Kihira Y, Miyamoto L, Ishizawa K, et al: Iron-induced skeletal muscle atrophy involves an Akt-forkhead box O3-E3 ubiquitin ligase-dependent pathway. J Trace Elem Med Biol. 35:66–76. 2016. View Article : Google Scholar : PubMed/NCBI
41	Reardon TF and Allen DG: Iron injections in mice increase skeletal muscle iron content, induce oxidative stress and reduce exercise performance. Exp Physiol. 94:720–730. 2009. View Article : Google Scholar : PubMed/NCBI
42	Kasztura M, Dzięgała M, Kobak K, Bania J, Mazur G, Banasiak W, Ponikowski P and Jankowska EA: Both iron excess and iron depletion impair viability of rat H9C2 cardiomyocytes and L6G8C5 myocytes. Kardiol Pol. 75:267–275. 2017. View Article : Google Scholar
43	Sequeira V, Nijenkamp LL, Regan JA and van der Velden J: The physiological role of cardiac cytoskeleton and its alterations in heart failure. Biochim Biophys Acta. 1838:700–722. 2014. View Article : Google Scholar
44	Lazarides E and Hubbard BD: Immunological characterization of the subunit of the 100 A filaments from muscle cells. Proc Natl Acad Sci USA. 73:4344–4348. 1976. View Article : Google Scholar : PubMed/NCBI
45	Koutakis P, Miserlis D, Myers SA, Kim JK, Zhu Z, Papoutsi E, Swanson SA, Haynatzki G, Ha DM, Carpenter LA, et al: Abnormal accumulation of desmin in gastrocnemius myofibers of patients with peripheral artery disease. J Histochem Cytochem. 63:256–269. 2015. View Article : Google Scholar : PubMed/NCBI
46	Russ DW and Grandy JS: Increased desmin expression in hindlimb muscles of aging rats. J Cachexia Sarcopenia Muscle. 2:175–180. 2011. View Article : Google Scholar : PubMed/NCBI
47	Walter PB, Knutson MD, Paler-Martinez A, Lee S, Xu Y, Viteri FE and Ames BN: Iron deficiency and iron excess damage mitochondria and mitochondrial DNA in rats. Proc Natl Acad Sci USA. 99:2264–2269. 2002. View Article : Google Scholar : PubMed/NCBI