Paes 2014 Abstract IOC 2014-04 Schroecken

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Paes MC, Saraiva FMS, Nogueira NP, Laranja GAT, Coelho MGP, Oliveira MF (2014) Heme increases mitochondrial membrane potential and ROS production in Trypanosoma cruzi epimastigotes. Mitochondr Physiol Network 19.02.

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Paes MC, Saraiva FMS, Nogueira NP, Laranja GAT, Coelho MGP, Oliveira MF (2014)

Event: MiPNet19.02 IOC88

Trypanosoma cruzi has a single mitochondrion, the main site of reactive oxygen species (ROS) production. Moreover, T. cruzi epimastigotes proliferate in the presence of heme, which induces ROS formation (Nogueira et al 2011; Lara et al 2007). Therefore, we evaluated heme effect upon mitochondrial ROS formation and mitochondrial membrane potential (ΔΨmt). For that, epimastigotes were incubated with DHE or TMRM with or without heme. After this, FCCP and antymicin A (Ama) were added. Mitochondrial ROS production and ΔΨmt were analyzed by flow cytometry. Our results showed that heme duplicated ROS production and induced a 4-fold increase of ΔΨmt. The FCCP addition reversed heme effects upon ROS generation and ΔΨm. Additionally, Ama induced a 2-fold increase of ROS production and 46% increment in ΔΨmt, while co-incubation with heme and AA presented a 3-fold increase upon ROS formation and increase ΔΨmt in 70%. In order to corroborate the involvement of heme in mitochondrial ROS, we incubated the parasites with heme, in the absence or in the presence of mitoTEMPO, a mitochondrial antioxidant. Our results showed that in the presence of this antioxidant greatly decreased heme induced ROS generation. Afterwards, we incubated epimastigotes with heme for 30 min and then, performed a substrate-uncoupler-inhibitor-tritation protocol with rotenone, succinate, ADP, cytocrome c, FCCP and Ama. We were able to detect a decrease in several states, mainly ROUTINE, OXPHOS and reserve capacity, compared to control cells. Finally, we evaluated epimastigotes proliferation with or without heme, H2O2, FCCP, Ama or mitoTEMPO. We observed that low concentrations of H2O2 increased proliferation, while higher concentrations showed deleterious effects upon the cells. FCCP and mitoTEMPO also reversed heme-induced proliferation, whereas, Ama promoted a tripanostatic effect. Taken together, our results strongly suggest that heme modulates T. cruzi mitochondrial physiology since it promotes mitochondrial ROS production, decreasing mitochondrial states, and enhances the ΔΨmt.

Keywords: Heme, ROS, Trypanosoma cruzi, mitochondria

O2k-Network Lab: BR Rio de Janeiro Paes MC, BR Rio de Janeiro Oliveira MF


Labels: MiParea: Respiration, mt-Membrane  Pathology: Infectious  Stress:Oxidative stress;RONS  Organism: Protists 

Preparation: Permeabilized cells, Oxidase;biochemical oxidation  Enzyme: Complex II;succinate dehydrogenase, Complex III, Uncoupling protein  Regulation: mt-Membrane potential, Oxygen kinetics, Redox state, Uncoupler  Coupling state: LEAK, OXPHOS, ET  Pathway: S, Other combinations  HRR: Oxygraph-2k 


Affiliations

Saraiva FMS (1), Nogueira NPA(1), Laranja GAT(1), Coelho MGP (1), Oliveira MF (2), and Paes MC (1)

(1) Instituto de Biologia Roberto Alcântara Gomes - Departamento de Bioquímica – Universidade do Estado do Rio de Janeiro – RJ - Brasil.

(2) Instituto de Bioquímica Médica – Universidade Federal do Rio de Janeiro – RJ- Brasil

Supported by CNPq, INCT-EM and FAPERJ