Hypoxia-Reoxygenation Induced Cardiac Mitochondrial Dysfunction

Year 2018, Volume: 71 Issue: 3, 17 - 27, 31.12.2018

Gül Şimşek , Hilmi Burak Kandilci Kandilci

Abstract

Objectives: Cellular response to low oxygen tension is altered by severity and duration of hypoxia. Although the subject has been studied extensively,
mechanisms leading to hypoxia-reoxygenation damage remain undefined. Here, we investigated the effect of long term continuous hypoxia (48
hours) on cardiac derived HL-1 cells, mainly the role of mitochondria in cellular energy and reactive oxygen species homeostasis.
Materials and Methods: In this study, mammalian atrium derived HL-1 cells were cultured either in long term hypoxia (48 hours, 1% O2) or in
normoxia (48 hours, 21% O2) conditions. Mitochondrial membrane potential and reactive oxygen species (ROS) level was measured using florescent
dyes in a confocal microscope. GAPDH protein levels were detected by western blotting in normoxic control and hypoxic cells.
Results: Present results demonstrate that, 48 hours of hypoxia did not alter baseline mitochondrial membrane potential and its oxidative respiration
capacity in cardiac HL-1 cells. The mitochondrial depolarization response to in reoxygenation period of oxygen deprived cells was slower in hypoxic
cells. In hypoxic cells, basal ROS levels were higher whereas hydrogen peroxide response was smaller when compared with the normoxic control
group. GAPDH protein levels were unaltered between groups.
Conclusion: Present results indicate that, persistent mitochondrial oxidation phosphorylation uncoupling may lead to an over production of ROS.

Keywords

HL-1 Cells , Hypoxia , Mitochondrial Membrane Potential , Reactive Oxygen Species

Project Number

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Hipoksi-Reoksijenasyon ile İndüklenen Kardiyak Mitokondriyal Disfonksiyon

Abstract

Amaç: Hücrelerin azalan oksijen konsantrasyonuna verdikleri yanıt hipoksinin şiddeti ve süresine göre değişiklik göstermektedir. Bu konuda oldukça fazla çalışma yapılmış olmasına karşın, hipoksi ile indüklenen fonksiyonel yanıtların altında yatan mekanizmalar büyük ölçüde aydınlatılamamıştır. Bu çalışmada, kardiyak kökenli HL-1 hücrelerinin, uzun süreli (48 saat) kesintisiz hipoksiye verdikleri yanıtta mitokondrinin hücresel enerji ve reaktif oksijen türevleri (ROS) dengesindeki rolü araştırılmıştır.

Gereç ve Yöntem: Bu çalışmada memeli atriyum kökenli hücre serisi HL-1 hücrelerinde uzun süreli hipokside (48 saat, %1 O2) ve normoksik (48 saat, %21 O2) şartlarda kültür edilmiştir. Normoksik kontrol ve hipoksik hücrelerden mitokondri membran potansiyeli ve ROS miktarı floresan boyalar ile konfokal mikroskopta ölçülmüş, GAPDH protein seviyeleri western blot yöntemi ile belirlenmiştir.

Bulgular: Sonuçlarımıza göre, kardiyak HL-1 hücrelerinde 48 saat hipoksi bazal mitokondri membran potansiyeli ve oksijenli solunum kapasitesini değiştirmedi. Bununla birlikte hipoksik hücrelerin reoksijenasyon sırasında ortamdan tekrar oksijen uzaklaştırılmasına olan mitokondriyal depolarizasyon yanıtları normoksik kontrol hücrelere göre yavaştı. Hipoksik hücrelerde bazal ROS miktarında artış gözlenirken, hidrojen peroksite olan yanıtlar normoksik kontrol grubuna göre azaldı. GAPDH protein seviyesinde gruplar arası bir fark saptanmadı.

Sonuç: Bu sonuçlar, uzun süreli hipoksi ile indüklenen mitokondriyal oksidatif fosforilasyon kenetindeki kalıcı disfonksiyonun hücresel ROS artışından
sorumlu olabileceğini düşündürmektedir.

Keywords

HL-1 Hücreleri , Hipoksi , Mitokondri Membran Potansiyeli , Reaktif Oksijen Türevleri

Ethical Statement

Etik Kurul Onayı: Etik kurul onayı alınmamıştır. Hasta Onayı: Hasta onayı alınmamıştır. Hakem Değerlendirmesi: Editörler kurulu dış tarafından değerlendirilmiştir.ında olan kişiler

Project Number

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Thanks

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References

• 1. Liu B, Tewari AK, Zhang L, et al. Proteomicanalysis of protein tyrosine nitration afteri schemiareperfusio ninjury:mitochondria as themajortarget. Biochim Biophys Acta 2009;1794:476-485.
• 2. Solaini G, Harris DA. Biochemicaldysfunction in heart mitochondria exposed to ischaemia and reperfusion. Biochem J. 2005;390:377-394.
• 3. Semenza GL.Hypoxia-induciblefactor 1: masterregulator of O2 homeostasis. CurrOpinGenet Dev. 1998;8:588-594.
• 4. Semenza GL, Roth PH, Fang HM, et al. Transcriptional regulation of genesencoding glycolytic enzymes by hypoxia-induciblefactor 1. J BiolChem 1994;269:23757-23763.
• 5. Semenza GL, Jiang BH, Leung SW, et al. Hypoxia response elements in thealdolase A, enolase 1, andlactatedehydrogenase A gene promoterscontain essential binding sitesfor hypoxia-inducible factor 1.J BiolChem 1996;271:32529-32537.
• 6. Ebert BL, Firth JD, Ratcliffe PJ. Hypoxia and mitochondrial inhibitorsregulate expression of glucose transporter-1 viadistinctCis-actingsequences. J Biol Chem 1995;270:29083-29089.
• 7. Turrens JF. Mitochondrial formation of reactive oxygen species. J Physiol 2003;552:335-344.
• 8. Papandreou I, Cairns RA, Fontana L, et al. HIF-1 mediates adaptation to hypoxia by actively downregulating mitochondrial oxygen consumption. Cell Metab. 2006;3:187-197.
• 9. Chandel NS, McClintock DS, Feliciano CE, et al. Reactive oxygen species generated at mitochondrial Complex III stabilize hypoxia-inducible factor-1alpha during hypoxia: a mechanism of O2 sensing. J Biol Chem. 2000;275:25130-25138.
• 10. Chandel NS, Maltepe E, Goldwasser E, et al. Mitochondrial reactive oxygen species trigger hypoxia-induced transcription. Proc Natl Acad Sci. 1998;95:11715-11720.
• 11. Baracca A, Chiaradonna F, Sgarbi G, et al. Mitochondrial Complex I decrease is responsible for bioenergetic dysfunction in K-ras transformed cells. Biochim Biophys Acta 2010;1797:314-323.
• 12. Şimşek G, Vaughan-Jones RD, Swietach P, et al. Recovery from hypoxiainducedinternalization of cardiacNa+/H+ exchanger 1 requires an adequate intracellular store of anti-oxidants. J Cell Physiol 2018.
• 13. Claycomb WC, Lanson NA, Stallworth BS, et al. HL‐1 cells: Acardiac muscle cell line that contracts and retains phenotypic characteristics of the adult cardiomyocyte. Proc Natl Acad Sci U S A. 1998;95:2979-2984.
• 14. Jezek P, Plecitá-Hlavatá L. Mitochondrial reticulum network dynamics in relation to oxidative stress, redox regulation, and hypoxia. Int J Biochem Cell Biol. 2009;41:1790-1804.
• 15. Skarka L, Ostadal B. Mitochondrial membra nepotential in cardiacmyocytes. Physiol Res 2002;51:425-434.
• 16. Halestrap AP, Pasdois P. The role of the mitochondrial permeabilitytransition pore in heartdisease. Biochim Biophys Acta. 2009;1787:1402-1415.
• 17. Sridharan V, Guichard J, Li CY, et al. O(2)-sensing signal cascade: clamping of O(2) respiration, reduced ATP utilization, and inducible fumarate respiration. Am J Physiol Cell Physiol. 2008;295:C29-37.
• 18. Ganitkevich V, Reil S, Schwethelm B, et al. Dynamic Responses of Single Cardiomyocytesto GradedIschemia Studiedby Oxygen Clamp in On-Chip Picochambers. Circ Res. 2006;99:165-171.
• 19. Chen Q, Moghaddas S, Hoppel CL, et al. Reversibleblockade of electron transport during ischemia protects mitochondria and decreases myocardial in jury followingreperfusion. J Pharmacol Exp Ther. 2006;319:1405-1412.
• 20. Chen CH, Budas GR, Churchill EN, et al. Activation of aldehyde dehydrogenase-2 reduces ischemic damag eto the hear. Science. 2008;321:1493-1495.
• 21. Lesnefsky EJ, Gudz TI, Migita CT, et al. Ischemic injury to mitochondrial electron transport in the aging heart: damage to the iron-sulfur protein subunit of electron transport complex III. Arch Biochem Biophys 2001;385:117-128.
• 22. Lesnefsky EJ, Slabe TJ, Stoll MS, et al. Myocardial ischemia selectively depletes cardiolipin in rabbit heart subsarcolemmal mitochondria. Am J Physiol Heart Circ Physiol. 2001;280:H2770-2778.
• 23. Görlach A, Dimova EY, Petry A, et al. Reactive oxygen species, nutrition, hypoxia and diseases: Problems solved? Redox Biology. 2015;6:372-385.