Paes 2014 Abstract IOC 2014-04 Schroecken

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: 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 (ΔΨm). For that, epimastigotes were incubated with DHE or TMRM with or without heme. After this, FCCP and antymicin A (AA) were added. Mitochondrial ROS production and ΔΨm were analyzed by flow cytometry. Our results showed that heme duplicated ROS production and induced a 4-fold increase of ΔΨm. The FCCP addition reversed heme effects upon ROS generation and ΔΨm. Additionally, AA induced a 2-fold increase of ROS production and 46% increment in ΔΨm, while co-incubation with heme and AA presented a 3-fold increase upon ROS formation and increase ΔΨm 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 an inhibitor-substrate-uncuppler-inhibitor-tritation protocol with rotenone, succinate, ADP, cytocrome C, FCCP and AA. 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, AA 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, AA 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 ΔΨm.

• 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 

Preparation: Permeabilized cells, Oxidase;biochemical oxidation  Enzyme: Complex II;succinate dehydrogenase, Complex III, Uncoupling protein  Regulation: mt-Membrane potential, O2"O2" is not in the list (Aerobic glycolysis, ADP, ATP, ATP production, AMP, Calcium, Coupling efficiency;uncoupling, Cyt c, Flux control, Inhibitor, ...) of allowed values for the "Respiration and regulation" property., Redox state, Uncoupler  Coupling state: ROUTINE, OXPHOS 

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